Pyrrole inhibitors of S-nitrosoglutathione reductase as therapeutic agents

ABSTRACT

The present invention is directed to inhibitors of S-nitrosoglutathione reductase (GSNOR), pharmaceutical compositions comprising such GSNOR inhibitors, and methods of making and using the same.

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national phase application ofPCT/US2009/053925, filed Aug. 14, 2009 (WO 2010/019905), entitled “NovelPyrrole Inhibitors of S-Nitrosoglutathione Reductase as TherapeuticAgents,” which claims priority to U.S. Provisional Application Ser. No.61/116,876, filed Nov. 21, 2008 and U.S. Provisional Application Ser.No. 61/089,313, filed Aug. 15, 2008. Each of these applications isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to novel pyrrole inhibitors ofS-nitrosoglutathione reductase, pharmaceutical compositions comprisingsuch inhibitors, and methods of making and using the same.

BACKGROUND OF THE INVENTION

The chemical compound nitric oxide is a gas with chemical formula NO. NOis one of the few gaseous signaling molecules known in biologicalsystems, and plays an important role in controlling various biologicalevents. For example, the endothelium uses NO to signal surroundingsmooth muscle in the walls of arterioles to relax, resulting invasodilation and increased blood flow to hypoxic tissues. NO is alsoinvolved in regulating smooth muscle proliferation, platelet function,neurotransmission, and plays a role in host defense. Although nitricoxide is highly reactive and has a lifetime of a few seconds, it canboth diffuse freely across membranes and bind to many molecular targets.These attributes make NO an ideal signaling molecule capable ofcontrolling biological events between adjacent cells and within cells.

NO is a free radical gas, which makes it reactive and unstable, thus NOis short lived in vivo, having a half life of 3-5 seconds underphysiologic conditions. In the presence of oxygen, NO can combine withthiols to generate a biologically important class of stable NO adductscalled S-nitrosothiols (SNO's). This stable pool of NO has beenpostulated to act as a source of bioactive NO and as such appears to becritically important in health and disease, given the centrality of NOin cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA,89:7674-7677 (1992)). Protein SNO's play broad roles in cardiovascular,respiratory, metabolic, gastrointestinal, immune and central nervoussystem function (Foster et al., 2003, Trends in Molecular MedicineVolume 9, Issue 4, April 2003, pages 160-168). One of the most studiedSNO's in biological systems is S-nitrosoglutathione (GSNO) (Gaston etal., Proc. Natl. Acad. Sci. USA 90:10957-10961 (1993)), an emerging keyregulator in NO signaling since it is an efficient trans-nitrosatingagent and appears to maintain an equilibrium with other S-nitrosatedproteins (Liu et al., 2001) within cells. Given this pivotal position inthe NO—SNO continuum, GSNO provides a therapeutically promising targetto consider when NO modulation is pharmacologically warranted.

In light of this understanding of GSNO as a key regulator of NOhomeostasis and cellular SNO levels, studies have focused on examiningendogenous production of GSNO and SNO proteins, which occurs downstreamfrom the production of the NO radical by the nitric oxide synthetase(NOS) enzymes. More recently there has been an increasing understandingof enzymatic catabolism of GSNO which has an important role in governingavailable concentrations of GSNO and consequently available NO andSNO's.

Central to this understanding of GSNO catabolism, researchers haverecently identified a highly conserved S-nitrosoglutathione reductase(GSNOR) (Jensen et al., Biochem J., 331:659-668 (1998); Liu et al.,Nature, 410:490-494 (2001)). GSNOR is also known asglutathione-dependent formaldehyde dehydrogenase (GS-FDH), alcoholdehydrogenase 3 (ADH-3) (Uotila and Koivusalo, Coenzymes and Cofactors,D. Dolphin, ed. pp. 517-551 (New York, John Wiley & Sons, 1989)), andalcohol dehydrogenase 5 (ADH-5). Importantly GSNOR shows greateractivity toward GSNO than other substrates (Jensen et al., 1998; Liu etal., 2001) and appears to mediate important protein and peptidedenitrosating activity in bacteria, plants, and animals. GSNOR appearsto be the major GSNO-metabolizing enzyme in eukaryotes (Liu et al.,2001). Thus, GSNO can accumulate in biological compartments where GSNORactivity is low or absent (e.g. airway lining fluid) (Gaston et al.,1993).

Yeast deficient in GSNOR accumulate S-nitrosylated proteins which arenot substrates of the enzyme, which is strongly suggestive that GSNOexists in equilibrium with SNO-proteins (Liu et al., 2001). Preciseenzymatic control over ambient levels of GSNO and thus SNO-proteinsraises the possibility that GSNO/GSNOR may play roles across a host ofphysiological and pathological functions including protection againstnitrosative stress wherein NO is produced in excess of physiologicneeds. Indeed, GSNO specifically has been implicated in physiologicprocesses ranging from the drive to breathe (Lipton et al., Nature,413:171-174 (2001)) to regulation of the cystic fibrosis transmembraneregulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001),to regulation of vascular tone, thrombosis and platelet function (deBelder et al., Cardiovasc Res. 1994 May; 28(5):691-4. (1994); Z.Kaposzta, A et al., Circulation; 106(24): 3057-3062, 2002) as well ashost defense (de Jesus-Berrios et al., Curr. Biol., 13:1963-1968(2003)). Other studies have found that GSNOR protects yeast cellsagainst nitrosative stress both in vitro (Liu et al., 2001) and in vivo(de Jesus-Berrios et al., 2003).

Collectively data suggest GSNOR as a primary physiological ligand forthe enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizesGSNO and consequently reduces available SNO's and NO in biologicalsystems (Liu et al., 2001), (Liu et al., Cell, (2004), 116(4), 617-628),and (Que et al., Science, 2005, 308, (5728):1618-1621). As such, thisenzyme plays a central role in regulating local and systemic bioactiveNO. Since perturbations in NO bioavailability has been linked to thepathogenesis of numerous disease states, including hypertension,atherosclerosis, thrombosis, asthma, gastrointestinal disorders,inflammation and cancer, agents that regulate GSNOR activity arecandidate therapeutic agents for treating diseases associated withnitric oxide imbalance.

Currently, there is a great need in the art for diagnostics,prophylaxis, ameliorations, and treatments for medical conditionsrelating to increased NO synthesis and/or increased NO bioactivity. Inaddition, there is a significant need for novel compounds, compositionsand methods for preventing, ameliorating, or reversing otherNO-associated disorders. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

The present invention provides novel pyrrole compounds useful asS-nitrosoglutathione reductase (“GSNOR”) inhibitors. The inventionencompasses pharmaceutically acceptable salts, prodrugs, and metabolitesof the described GSNOR inhibitors. Also encompassed by the invention arepharmaceutical compositions comprising at least one GSNOR inhibitor andat least one pharmaceutically acceptable carrier.

The compositions of the present invention can be prepared in anysuitable pharmaceutically acceptable dosage form.

The present invention provides a method for inhibitingS-nitrosoglutathione reductase in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The present invention also provides a method of treating a disorderameliorated by NO donor therapy in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug, or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The present invention also provides a method of treating a cellproliferative disorder in a subject in need thereof. Such a methodcomprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug, or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The methods of the invention encompass administration with one or moresecondary active agents. Such administration can be sequential or in acombination composition.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publiclyavailable publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control.

Both the foregoing summary and the following detailed description areexemplary and explanatory and are intended to provide further details ofthe compositions and methods as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Overview of theInvention

Until recently, S-nitrosoglutathione reductase (GSNOR) was known tooxidize the formaldehyde glutathione adduct, S-hydroxymethylglutathione.GSNOR has since been identified in a variety of bacteria, yeasts, plantsand animals and is well conserved. The proteins from E. coli, S.cerevisiae and mouse macrophages share over 60% amino acid sequenceidentity. GSNOR activity (i.e., decomposition of S-nitrosoglutathionewhen NADH is present as a required cofactor) has been detected in E.coli, in mouse macrophages, in mouse endothelial cells, in mouse smoothmuscle cells, in yeasts, and in human HeLa, epithelial and monocytecells. Human GSNOR nucleotide and amino acid sequence information can beobtained from the National Center for Biotechnology Information (NCBI)databases under Accession Nos. M29872, NM_(—)000671. Mouse GSNORnucleotide and amino acid sequence information can be obtained from NCBIdatabases under Accession Nos. NM_(—)007410. In the nucleotide sequence,the start site and stop site are underlined. CDS designates codingsequence. SNP designates single nucleotide polymorphism. Other relatedGSNOR nucleotide and amino acid sequences, including those of otherspecies, can be found in U.S. Patent Application 2005/0014697.

In accord with the present invention, GSNOR has been shown to functionin vivo and in vitro to metabolize S-nitrosoglutathione (GSNO) andprotein S-nitrosothiols (SNOs) to modulate NO bioactivity, bycontrolling the intracellular levels of low mass NO donor compounds andpreventing protein nitrosylation from reaching toxic levels.

Based on this, it follows that inhibition of this enzyme potentiatesbioactivity in all diseases in which NO donor therapy is indicated,inhibits the proliferation of pathologically proliferating cells, andincreases NO bioactivity in diseases where this is beneficial.

The present invention provides pharmaceutical agents that are potentinhibitors of GSNOR. In particular, provided are substituted pyrroleanalogs that are inhibitors of GSNOR having the structures depictedbelow (Formula I), or a pharmaceutically acceptable salt, stereoisomer,or prodrug thereof.

Tri-substituted pyrrole analogs are potent inhibitors of GSNOR. As usedin this context, the term “analog” refers to a compound having similarchemical structure and function as compounds of formula I that retainsthe pyrrole ring.

Some pyrrole analogs of the invention can also exist in various isomericforms, including configurational, geometric and conformational isomers,as well as existing in various tautomeric forms, particularly those thatdiffer in the point of attachment of a hydrogen atom. As used herein,the term “isomer” is intended to encompass all isomeric forms of acompound including tautomeric forms of the compound.

Illustrative compounds having asymmetric centers can exist in differentenantiomeric and diastereomeric forms. A compound can exist in the formof an optical isomer or a diastereomer. Accordingly, the inventionencompasses compounds in the forms of their optical isomers,diastereomers and mixtures thereof, including racemic mixtures.

It should be noted that if there is a discrepancy between a depictedstructure and a name given to that structure, the depicted structurecontrols. In addition, if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold, wedged,or dashed lines, the structure or portion of the structure is to beinterpreted as encompassing all stereoisomers of the described compound.

In accordance with the invention, the levels of the S-nitrosoglutathionereductase in the biological sample can be determined by the methodsdescribed in U.S. Patent Application Publication No. 2005/0014697. Theterm “biological sample” includes, but is not limited to, samples ofblood (e.g., serum, plasma, or whole blood), urine, saliva, sweat,breast milk, vaginal secretions, semen, hair follicles, skin, teeth,bones, nails, or other secretions, body fluids, tissues, or cells.

B. Definitions

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

The term “acyl” includes compounds and moieties that contain the acetylradical (CH₃CO—) or a carbonyl group to which a straight or branchedchain lower alkyl residue is attached.

The term “alkyl” as used herein refers to a straight or branched chain,saturated hydrocarbon having the indicated number of carbon atoms. Forexample, (C₁-C₆) alkyl is meant to include, but is not limited tomethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. An alkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein.

The term “alkenyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one double bond. Examples of a (C₂-C₈) alkenyl group include,but are not limited to, ethylene, propylene, 1-butylene, 2-butylene,isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene,2-hexene, 3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene,isoheptene, 1-octene, 2-octene, 3-octene, 4-octene, and isooctene. Analkenyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkynyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one triple bond. Examples of a (C₂-C₈) alkynyl group include,but are not limited to, acetylene, propyne, 1-butyne, 2-butyne,1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne,2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. Analkynyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkoxy” as used herein refers to an —O-alkyl group having theindicated number of carbon atoms. For example, a (C₁-C₆) alkoxy groupincludes —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl,—O-sec-butyl, —O-tert-butyl, —O-pentyl, —O-isopentyl, —O-neopentyl,—O-hexyl, —O-isohexyl, and —O-neohexyl.

The term “aminoalkyl” as used herein, refers to an alkyl group(typically one to six carbon atoms) wherein one or more of the C₁-C₆alkyl group's hydrogen atoms is replaced with an amine of formula—N(R^(c))₂, wherein each occurrence of R^(c) is independently —H or(C₁-C₆) alkyl. Examples of aminoalkyl groups include, but are notlimited to, —CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂N(CH₃)₂,t-butylaminomethyl, isopropylaminomethyl and the like.

The term “aryl” as used herein refers to a 5- to 14-membered monocyclic,bicyclic or tricyclic aromatic ring system. Examples of an aryl groupinclude phenyl and naphthyl. An aryl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow. Examples of aryl groups include phenyl or aryl heterocycles suchas, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

The term “carbonyl” or “carboxy” or “carboxyl” includes compounds andmoieties which contain a carbon connected with a double bond to anoxygen atom. Examples of moieties containing a carbonyl include, but arenot limited to, aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc.

The term “C_(m)-C_(n)” means “m” number of carbon atoms to “n” number ofcarbon atoms. For example, the term “C₁-C₆” means one to six carbonatoms (C₁, C₂, C₃, C₄, C₅ or C₆). The term “C₂-C₆” includes two to sixcarbon atoms (C₂, C₃, C₄, C₅ or C₆). The term “C₃-C₆” includes three tosix carbon atoms (C₃, C₄, C₅ or C₆).

The term “cycloalkyl” as used herein refers to a 3- to 14-memberedsaturated or unsaturated non-aromatic monocyclic, bicyclic or tricyclichydrocarbon ring system. Included in this class are cycloalkyl groupswhich are fused to a benzene ring. Representative cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl,cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, cycloheptyl, cycloheptenyl, 1,3-cycloheptadienyl,1,4-cycloheptadienyl, -1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctenyl, 1,3-cyclooctadienyl, 1,4-cyclooctadienyl,-1,3,5-cyclooctatrienyl, decahydronaphthalene, octahydronaphthalene,hexahydronaphthalene, octahydroindene, hexahydroindene, tetrahydroinden,decahydrobenzocycloheptene, octahydrobenzocycloheptene,hexahydrobenzocycloheptene, tetrahydrobenzocyclopheptene,dodecahydroheptalene, decahydroheptalene, octahydroheptalene,hexahydroheptalene, and tetrahydroheptalene,(1s,3s)-bicyclo[1.1.0]butane, bicyclo[1.1.1]pentane,bicyclo[2.1.1]hexane, Bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane,bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[3.3.1]nonane,bicyclo[3.3.2]decane, bicyclo[3.3.]undecane, bicyclo[4.2.2]decane,bicyclo[4.3.1]decane. A cycloalkyl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.

The term “haloalkyl” as used herein, refers to a C₁-C₆ alkyl groupwherein from one or more of the C₁-C₆ alkyl group's hydrogen atom isreplaced with a halogen atom, which can be the same or different.Examples of haloalkyl groups include, but are not limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl,pentachloroethyl, and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “heteroalkyl” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chainalkyl, or combinations thereof, consisting of carbon atoms and from oneto three heteroatoms selected from the group consisting of O, N and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. The heteroatom(s)O, N and S can be placed at any position of the heteroalkyl group.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, and—CH₂—CH═N—OCH₃. Up to two heteroatoms can be consecutive, such as, forexample, —CH₂—NH—OCH₃. When a prefix such as (C₂-C₈) is used to refer toa heteroalkyl group, the number of carbons (2 to 8, in this example) ismeant to include the heteroatoms as well. For example, a C₂-heteroalkylgroup is meant to include, for example, —CH₂OH (one carbon atom and oneheteroatom replacing a carbon atom) and —CH₂SH.

To further illustrate the definition of a heteroalkyl group, where theheteroatom is oxygen, a heteroalkyl group can be an oxyalkyl group. Forinstance, (C₂-C₅) oxyalkyl is meant to include, for example —CH₂—O—CH₃(a C₃-oxyalkyl group with two carbon atoms and one oxygen replacing acarbon atom), —CH₂CH₂CH₂CH₂OH, —OCH₂CH₂OCH₂CH₂OH, —OCH₂CH(OH)CH₂OH, andthe like.

The term “heteroaryl” as used herein refers to an aromatic heterocyclering of 5 to 14 members and having at least one heteroatom selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom,including monocyclic, bicyclic, and tricyclic ring systems.Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl,pyridyl, furyl, benzofuranyl, thienyl, benzothienyl, quinolinyl,pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, quinoxalinyland oxazolyl. A heteroaryl group can be unsubstituted or optionallysubstituted with one or more substituents as described herein below.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), and sulfur (S).

As used herein, the term “heterocycle” refers to 3- to 14-membered ringsystems which are either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms canbe optionally oxidized, and the nitrogen heteroatom can be optionallyquaternized, including, including monocyclic, bicyclic, and tricyclicring systems. The bicyclic and tricyclic ring systems may encompass aheterocycle or heteroaryl fused to a benzene ring. The heterocycle canbe attached via any heteroatom or carbon atom, where chemicallyacceptable. Heterocycles include heteroaryls as defined above.Representative examples of heterocycles include, but are not limited to,aziridinyl, oxiranyl, thiiranyl, triazolyl, tetrazolyl, azirinyl,diaziridinyl, diazirinyl, oxaziridinyl, azetidinyl, azetidinonyl,oxetanyl, thietanyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl,oxazinyl, thiazinyl, diazinyl, dioxanyl, triazinyl, tetrazinyl,imidazolyl, tetrazolyl, pyrrolidinyl, isoxazolyl, furanyl, furazanyl,pyridinyl, oxazolyl, benzoxazolyl, benzisoxazolyl, thiazolyl,benzthiazolyl, thienyl, pyrazolyl, triazolyl, pyrimidinyl,benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl, benzotriazolyl,benzoxazolyl, benzisoxazolyl, purinyl, indolyl, isoquinolinyl,quinolinyl and quinazolinyl. A heterocycle group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow.

The term “heterocycloalkyl” by itself or in combination with otherterms, represents, unless otherwise stated, cyclic versions of“heteroalkyl.” Additionally, a heteroatom can occupy the position atwhich the heterocycle is attached to the remainder of the molecule.Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like.

The term “hydroxyalkyl” as used herein, refers to an alkyl group havingthe indicated number of carbon atoms wherein one or more of the hydrogenatoms in the alkyl group is replaced with an —OH group. Examples ofhydroxyalkyl groups include, but are not limited to, —CH₂OH, —CH₂CH₂OH,—CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂CH₂OH, and branched versions thereof.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O—.

As used herein and unless otherwise indicated, the term “stereoisomer”means one stereoisomer of a compound that is substantially free of otherstereoisomers of that compound. For example, a stereomerically purecompound having one chiral center will be substantially free of theopposite enantiomer of the compound. A stereomerically pure compoundhaving two chiral centers will be substantially free of otherdiastereomers of the compound. In some embodiments, a stereomericallypure compound comprises greater than about 80% by weight of onestereoisomer of the compound and less than about 20% by weight of otherstereoisomers of the compound, for example greater than about 90% byweight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “modulate” is meant to refer to an increase or decreasethe levels of a peptide or polypeptide, or to increase or decrease thestability or activity of a peptide or a polypeptide. The term “inhibit”is meant to refer to a decrease in the levels of a peptide or apolypeptide or to decrease in the stability or activity of a peptide ora polypeptide. In preferred embodiments, the peptide which is modulatedor inhibited is S-nitrosoglutathione (GSNO) or protein S-nitrosothiols(SNOs).

As used here, the terms “nitric oxide” and “NO” encompass unchargednitric oxide and charged nitric oxide species, particularly includingnitrosonium ion (NO⁺) and nitroxyl ion (NO⁻). The reactive form ofnitric oxide can be provided by gaseous nitric oxide. Compounds havingthe structure X—NO_(y) wherein X is a nitric oxide releasing, deliveringor transferring moiety, including any and all such compounds whichprovide nitric oxide to its intended site of action in a form active fortheir intended purpose, and Y is 1 or 2.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered and includes, but is not limitedto such sterile liquids as water and oils.

A “pharmaceutically acceptable salt” or “salt” of a GSNOR inhibitor is aproduct of the disclosed compound that contains an ionic bond, and istypically produced by reacting the disclosed compound with either anacid or a base, suitable for administering to a subject. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

A “pharmaceutical composition” is a formulation comprising the disclosedcompounds in a form suitable for administration to a subject. Apharmaceutical composition of the invention is preferably formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include, but are not limited to, oral andparenteral, e.g., intravenous, intradermal, subcutaneous, inhalation,topical, transdermal, transmucosal, and rectal administration.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N, or N═N).

Substituents for the groups referred to as alkyl, heteroalkyl, alkylene,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl can be selected from a variety of groups including—OR^(d), ═O, ═NR^(d)′, ═N—OR^(d)′, —NR^(d)′R^(d)″, —SR^(d)′, -halo,—SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′, —C(O)R^(d)′, —CO₂R^(d)′,—CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)′″C(O)NR^(d)′R^(d)″, —NR^(d)′″SO₂NR^(d)′R^(d)″,—NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH, —NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′,—S(O)R^(d)′, —SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN and—NO₂, in a number ranging from zero to three, with those groups havingzero, one or two substituents being exemplary.

R^(d)′, R^(d)″ and R^(d)′″ each independently refer to hydrogen,unsubstituted (C₁-C₈)alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl and aryl substituted with one to three substituentsselected from -halo, unsubstituted alkyl, unsubstituted alkoxy,unsubstituted thioalkoxy and unsubstituted aryl (C₁-C₄)alkyl. WhenR^(d)′ and R^(d)″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6- or 7-membered ring. Forexample, —NR^(d)′R^(d)″ can represent 1-pyrrolidinyl or 4-morpholinyl.

Typically, an alkyl or heteroalkyl group will have from zero to threesubstituents, with those groups having two or fewer substituents beingexemplary of the present invention. An alkyl or heteroalkyl radical canbe unsubstituted or monosubstituted. In some embodiments, an alkyl orheteroalkyl radical will be unsubstituted.

Exemplary substituents for the alkyl and heteroalkyl radicals includebut are not limited to —OR″, ═O, ═NR^(d), ═N—OR^(d)′, —NR^(d)′R^(d)″,—SR^(d)′, -halo, —SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′, —C(O)R^(d)′,—CO₂R^(d)′, —CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)′″C(O)NR^(d)′R^(d)″, —NR^(d)″SO₂NR^(d)′R^(d)″, —NR^(d)″CO₂R^(d)′,—NHC(NH₂)═NH, —NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′″S(O)R^(d)′,—SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN and —NO₂, whereR^(d)′, R^(d)″ and R^(d)′″ are as defined above. Typical substituentscan be selected from: —OR^(d), ═O, —NR^(d)′R^(d)″, -halo, —OC(O)R^(d)′,—CO₂R^(d)′, —C(O)NR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)″CO₂R^(d)′, —NR^(d)′SO₂NR^(d)′R^(d)″, —SO₂R^(d)′,—SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′—CN and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: -halo, —OR^(e)′, —OC(O)R^(e)′, —NR′R^(e)″, —SR^(e)′,—R^(e)′, —CN, —NO₂, —CO₂R^(e)′, —C(O)NR′R^(e)″, —C(O)R^(e)′,—OC(O)NR′R^(e)″, —NR^(e)″C(O)R^(e)′, —NR^(e)″CO₂R^(e)′,—NR^(e)′″C(O)NR^(e)′R^(e)″, —NR^(e)′″SO₂NR^(e)′R^(e)″, —NHC(NH₂)═NH,—NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R^(e)′, —SO₂R^(e)′, —SO₂NR′R^(e)″,—NR^(e)″SO₂R^(e)′, —N₃, —CH(Ph)₂, perfluoroalkoxy andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system.

R^(e)′, R^(e)″ and R^(e)′″ are independently selected from hydrogen,unsubstituted (C₁-C₈) alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, unsubstituted heteroaryl, unsubstituted aryl(C₁-C₄)alkyl and unsubstituted aryloxy(C₁-C₄) alkyl. Typically, an aryl orheteroaryl group will have from zero to three substituents, with thosegroups having two or fewer substituents being exemplary in the presentinvention. In one embodiment of the invention, an aryl or heteroarylgroup will be unsubstituted or monosubstituted. In another embodiment,an aryl or heteroaryl group will be unsubstituted.

Two of the substituents on adjacent atoms of an aryl or heteroaryl ringin an aryl or heteroaryl group as described herein may optionally bereplaced with a substituent of the formula -T-C(O)—(CH₂)_(q)—U—, whereinT and U are independently —NH—, —O—, —CH₂— or a single bond, and q is aninteger of from 0 to 2. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -J-(CH₂)_(r)—K—, wherein J and K areindependently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR^(f)′— ora single bond, and r is an integer of from 1 to 3. One of the singlebonds of the new ring so formed may optionally be replaced with a doublebond. Alternatively, two of the substituents on adjacent atoms of thearyl or heteroaryl ring may optionally be replaced with a substituent ofthe formula —(CH₂), —X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR^(f)′—, —S—, —S(O)—, —S(O)₂—,or —S(O)₂NR^(a)′—. The substituent R^(f)′ in —NR^(f)′— and—S(O)₂NR^(f)′— is selected from hydrogen or unsubstituted (C₁-C₆) alkyl.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

As used herein the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder to be prevented, reduced, or treated as described herein. Thephrase “therapeutically effective amount” as it relates to the GSNORinhibitors of the present invention shall mean the GSNOR inhibitordosage that provides the specific pharmacological response for which theGSNOR inhibitor is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a GSNOR inhibitor that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

C. S-Nitrosoglutathione Reductase Inhibitors 1. Inventive Compounds

In one of its aspects the present invention provides a compound having astructure shown in Formula I, or a pharmaceutically acceptable salt,stereoisomer, or prodrug thereof:

whereinAr₁ and Ar₂ are independently selected from the group consisting ofaryl, substituted aryl, heteroaryl and substituted heteroaryl;X is selected from the group consisting of C or N;Y is C when X is N, and N when X is C; andn is 0-3.

In a further aspect of the invention, suitable identity for Ar₁includes, but is not limited to, substituted phenyl; and suitableidentities for Ar₂ includes, but are not limited to, the groupconsisting of phenyl, substituted phenyl, thiophen-yl, substitutedthiophen-yl, pyridinyl, substituted pyridinyl, thiazolyl, substitutedthiazolyl, bicyclic aryl, substituted bicyclic aryl, bicyclicheteroaryl, substituted bicyclic heteroaryl.

In a further aspect of the invention, a suitable identity of Ar₁includes, but is not limited to,

whereinR₁ is selected from the group consisting of hydrogen, halogen, hydroxyl,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, CF₃, carbamoyl, C₁-C₆alkylcarbamoyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆alkoxyl, and C₃-C₆ cycloalkoxyl;R₂ is selected from the group consisting of halogen, hydroxyl,carbamoyl, substituted carbamoyl, C₁-C₆ alkylcarbamoyl, sulfamoyl, C₁-C₆alkylsulfamoyl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, amino, CF₃,carboxyl, ureido, sulfamoylamino, C₁-C₆ sulfonamido, 2-amino-2-oxoethyl,C₁-C₆ alkylamino, C₁-C₆ dialkylamino, arylamino, heteroarylamino, C₁-C₆alkoxyl, C₃-C₆ cycloalkoxyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl;R₃ is selected from the group consisting of hydrogen, hydroxyl, halogen,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, carbamoyl, C₁-C₆alkylcarbamoyl, sulfamoyl, C₁-C₆ alkyl sulfamoyl, amino, C₁-C₆alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkoxyl, and C₃-C₆ cycloalkoxyl.

In a further aspect of the invention, suitable identities for R₁, R₂, R₃include, but are not limited to:

R₁ is selected from the group consisting of hydrogen and methyl;

R₂ is selected from the group consisting of hydroxyl, carboxyl,carbamoyl, methylsulfonamido, and tert-butyl-carboxy; and

R₃ is hydrogen.

In a further aspect of the invention, suitable identities for Ar₂include, but are not limited to, phenyl, 4-chlorophenyl, 3-chlorophenyl,4-chloro-2-methoxyphenyl, 4-bromophenyl, 4-bromo-2-methoxyphenyl,3-bromophenyl, 4-fluorophenyl, 3-fluorophenyl, 4-hydroxyphenyl,4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl,4-chlorothiophen-2-yl, 5-chlorothiophen-2-yl, 3-bromothiophen-2-yl,4-bromothiophen-2-yl, 5-bromothiopheny-2-yl, 5-bromothiophen-3-yl,

whereinR₄ is selected from the group consisting of hydrogen, methyl, chloro,fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl, dimethylamino,amino, formamido, and trifluoromethylR₅ is selected from the group consisting of hydrogen, methyl, and ethyl.

In a further aspect of the invention, suitable identities for Ar₂include, but are not limited to, phenyl, 4-chloro-2-methoxyphenyl,4-bromophenyl, 4-bromo-2-methoxyphenyl, 4-methoxyphenyl,4-(1H-imidazol-1-yl)phenyl, 4-(2-methyl-1H-imidazol-1-yl)phenyl, and5-(2-methyl-1H-imidazol-1-yl)thiophen-2-yl.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

In certain embodiments of the invention, Compound 1 may be excluded.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Alltautomers of shown or described compounds are also considered to be partof the present invention.

It is to be understood that isomers arising from such asymmetry (e.g.,all enantiomers and diastereomers) are included within the scope of theinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Alkenes can include either the E- or Z-geometry,where appropriate.

2. Representative GSNOR Inhibitors

Table 1 below lists representative novel pyrrole analogs of Formula Iuseful as GSNOR inhibitors of the invention. The synthetic methods thatcan be used to prepare each compound, identified in Table 1 (i.e. Scheme#1, Scheme #2, etc.), are detailed below in Example 1. In some cases, ifthe intermediate of a scheme is not commercially available, then acorresponding method (called Intermediate A, Intermediate B, etc.)describes the synthesis of that starting material or intermediate. Table1 provides Scheme #, defines starting materials shown in Schemes, andwhere necessary provides an Intermediate reference. Supporting massspectrometry data for each compound is also included in Table 1. GSNORinhibitor activity was determined by the assay described in Example 2and IC₅₀ values were obtained. GSNOR inhibitor compounds 1-24 of Table 1had an IC₅₀ of about <100 μM. GSNOR inhibitor compounds 1, 4-6, and 8-23of Table 1 had an IC₅₀ of about less than 5.0 μM and GSNOR inhibitorcompounds 1, 5, 8-9, 11-12, and 14-22 of Table 1 had an IC₅₀ of aboutless than 1.0 μM.

TABLE 1 Molec- Chemical ular Mass # Structure Compound name formulaweight Spec Synthesis 1

4-(2-(2-(1H-tetrazol-5- yl)ethyl)- 5-phenyl-1H-pyrrol-1- yl)benzamideC20H18N6O 358.4 359.3 Scheme 1, 1H where R2 = phenyl 2

4-(2-(2-(1H-tetrazol-5- yl)ethyl)- 5-phenyl-1H-pyrrol-1- yl)benzoic acidC20H17N5O2 359.4 360.3 Scheme 1, 1 G where R2 = phenyl 3

tert-butyl 4-(2-(2-(1H- tetrazol-5-yl)ethyl)-5- phenyl-1H-pyrrol-1-yl)benzoate C24H25N5O2 415.5 416.3 Scheme 1, 1F where R2 = phenyl 4

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4- methoxyphenyl)-1H-pyrrol-1-yl)-3- methylbenzamide C22H22N6O2 402.4 403.2 Scheme 2, R2 = 4-methoxyphenyl/ Intermediate C 5

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4- bromophenyl)-1H-pyrrol-1-yl)phenol C19H16BrN5O 410.3 412.1 Scheme 3, R2 = 4-bromophenyl/ Intermediate D 6

4-(2-((1H-tetrazol-5- yl)methyl)-5-(4- bromophenyl)-1H-pyrrol-1-yl)phenol C18H14BrN5O 396.2 396.0 Scheme 4, R2 = 4-bromophenyl, Intermediate A 7

4-(2-((1H-tetrazol-5- yl)methyl)-5-(4- methoxyphenyl)-1H-pyrrol-1-yl)phenol C19H17N5O2 347.4 348.0 Scheme 4, R2 = 4-methoxyphenyl, Intermediate B 8

4-(2-(4-(1H-imidazol-1- yl)phenyl)-5-(2-(1H- tetrazol-5-yl)ethyl)-1H-pyrrol-1-yl)phenol C22H19N7O 397.4 398.2 Scheme 6, R1 = 4-hydroxyphenyl, where 6A is Example #5 in Table 9

4-(2-((1H-tetrazol-5- yl)methyl)- 5-(4-bromophenyl)-1H- pyrrol-1-yl)-3-methylbenzamide C20H17BrN6O 437.3 437.0, 439.0 Scheme 5, R2 = 4-bromophenyl/ Intermediate A 10

4-(2-((1H-tetrazol-5- yl)methyl)-5-(4- methoxyphenyl)-1H-pyrrol-1-yl)-3- methylbenzamide C21H20N6O2 388.4 389.2 Scheme 5, R2 = 4-methoxyphenyl/ Intermediate B 11

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4- bromophenyl)-1H- pyrrol-1-yl)-3-methylbenzamide C21H19BrN6O 451.3 451.1 Scheme 2, R2 = 4- bromophenyl/Intermediate D 12

4-(2-(4-(1H-imidazol-1- yl)phenyl)-5-((1H- tetrazol-5-yl)methyl)-1H-pyrrol-1-yl)phenol C21H17N7O 383.4 384.0 Scheme 6, R1 = 4-hydroxyphenyl, where 6A is Example #5 in Table (reaction was heated at150° C. for 1 hr/ microwave) 13

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4- methoxyphenyl)-1H-pyrrol-1-yl)phenol C20H19N5O2 361.4 362.1 Scheme 3, R2 = 4-methoxyphenyl/ Intermediate C 14

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4-chloro-2- methoxyphenyl)-1H-pyrrol-1-yl)phenol C20H18ClN5O2 395.8 396.1 Scheme 3, R2 =4-chloro-2- methoxyphenyl/ Intermediate F 15

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4-bromo-2- methoxyphenyl)-1H-pyrrol-1-yl)phenol C20H18BrN5O2 440.3 439.8, 441.9 Scheme 3, R2 =4-bromo-2- methoxyphenyl/ Intermediate E 16

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4-chloro-2- methoxyphenyl)-1H-pyrrol-1-yl)-3- methylbenzamide C22H21ClN6O2 436.9 436.9 Scheme 2, R2= 4-chloro-2- methoxyphenyl/ Intermediate F 17

4-(2-(2-(1H-tetrazol-5- yl)ethyl)-5-(4-bromo-2- methoxyphenyl)-1H-pyrrol-1-yl)-3- methylbenzamide C22H21BrN6O2 481.3 483.1 Scheme 2, R2= 4-bromo-2- methoxyphenyl/ Intermediate E 18

4-(2-((1H-tetrazol-5- yl)methyl)-5-(4-(2- methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-1- yl)phenol C22H19N7O 397.4 397.9 Scheme 7, R1 =4- hydroxyphenyl, where 7A is Example #6 in this Table 19

4-(2-((1H-tetrazol-5- yl)methyl)-5-(5-(2- methyl-1H-imidazol-1-yl)thiophen-2-yl)-1H- pyrrol-1-yl)-3- methylbenzamide C22H20N8OS 444.5444.9 Scheme 8, R1 = 4-carbamoyl- 2-methyl-phenyl 20

4-(2-((1H-tetrazol-5- yl)methyl)-5-(5-(2- methyl-1H-imidazol-1-yl)thiophen-2-yl)-1H- pyrrol-1-yl)phenol C20H17N7OS 403.5 403.9 Scheme4, R2 = 5- bromothiophen- 2-yl, then followed Scheme 7 21

4-(2-((1H-tetrazol-5- yl)methyl)-5-(4-(2- methyl-1H-imidazol-1-yl)phenyl)-1H- pyrrol-1-yl)-3- methylbenzamide C24H22N8O 438.5 439.1Scheme 7, R1 = 4- carbamoyl-2- methylphenyl, where 7A is Example #9 inthis Table 22

N-(4-(2-((1H-tetrazol- 5-yl)methyl)-5-(5-(2- methyl-1H-imidazol-1-yl)thiophen-2-yl)- 1H-pyrrol-1-yl)-3- methylphenyl) methanesulfonamideC22H22N8O2S2 494.6 495.0 Scheme 7, R1 = 2-methyl 4- (methylsulfonamido)phenyl, where 7A is Example #9 in this Table 23

4-(1-(2-(1H-tetrazol-5- yl)ethyl)-3-(4- methoxyphenyl)-1H-pyrrol-2-yl)-3- methylbenzamide C22H22N6O2 402.4 403.1 Scheme 9, R =2-(1H- tetrazol-5-yl)ethyl 24

4-(1-((1H-tetrazol-5- yl)methyl)-3-(4- methoxyphenyl)-1H-pyrrol-2-yl)-3- methylbenzamide C21H20N6O2 388.4 389.2 Scheme 9, R =(1H-tetrazol-5- yl)methyl

D. Pharmaceutical Compositions Comprising a GSNOR Inhibitor

The invention encompasses pharmaceutical compositions comprising atleast one GSNOR inhibitor described herein and at least onepharmaceutically acceptable carrier. Suitable carriers are described in“Remington: The Science and Practice, Twentieth Edition,” published byLippincott Williams & Wilkins, which is incorporated herein byreference. Pharmaceutical compositions according to the invention mayalso comprise one or more non-GSNOR inhibitor active agents.

The pharmaceutical compositions of the invention can comprise novelGSNOR inhibitors described herein, the pharmaceutical compositions cancomprise known compounds which previously were not know to have GSNORinhibitor activity, or a combination thereof.

The GSNOR inhibitors can be utilized in any pharmaceutically acceptabledosage form, including but not limited to injectable dosage forms,liquid dispersions, gels, aerosols, ointments, creams, lyophilizedformulations, dry powders, tablets, capsules, controlled releaseformulations, fast melt formulations, delayed release formulations,extended release formulations, pulsatile release formulations, mixedimmediate release and controlled release formulations, etc.Specifically, the GSNOR inhibitors described herein can be formulated:(a) for administration selected from the group consisting of oral,pulmonary, intravenous, intra-arterial, intrathecal, intra-articular,rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal,intraperitoneal, local, buccal, nasal, and topical administration; (b)into a dosage form selected from the group consisting of liquiddispersions, gels, aerosols, ointments, creams, tablets, sachets andcapsules; (c) into a dosage form selected from the group consisting oflyophilized formulations, dry powders, fast melt formulations,controlled release formulations, delayed release formulations, extendedrelease formulations, pulsatile release formulations, and mixedimmediate release and controlled release formulations; or (d) anycombination thereof.

For respiratory infections, an inhalation formulation can be used toachieve high local concentrations. Formulations suitable for inhalationinclude dry power or aerosolized or vaporized solutions, dispersions, orsuspensions capable of being dispensed by an inhaler or nebulizer intothe endobronchial or nasal cavity of infected patients to treat upperand lower respiratory bacterial infections.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citratesor phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may comprisesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The pharmaceutical composition should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent (e.g., GSNOR inhibitor) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating at least one GSNOR inhibitor into a sterilevehicle that contains a basic dispersion medium and any other requiredingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, both of which yield a powder of theGSNOR inhibitor plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the GSNOR inhibitor can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In one embodiment, the GSNOR inhibitors are prepared with carriers thatwill protect against rapid elimination from the body. For example, acontrolled release formulation can be used, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of the GSNOR inhibitors may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils, such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate, triglycerides, or liposomes. Non-lipidpolycationic amino polymers may also be used for delivery. Optionally,the suspension may also include suitable stabilizers or agents toincrease the solubility of the compounds and allow for the preparationof highly concentrated solutions.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of GSNORinhibitor calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the GSNOR inhibitor and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active agent for thetreatment of individuals.

Pharmaceutical compositions according to the invention comprising atleast one GSNOR inhibitor can comprise one or more pharmaceuticalexcipients. Examples of such excipients include, but are not limited tobinding agents, filling agents, lubricating agents, suspending agents,sweeteners, flavoring agents, preservatives, buffers, wetting agents,disintegrants, effervescent agents, and other excipients. Suchexcipients are known in the art. Exemplary excipients include: (1)binding agents which include various celluloses and cross-linkedpolyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101and Avicel® PH102, silicified microcrystalline cellulose (ProSolvSMCC™), gum tragacanth and gelatin; (2) filling agents such as variousstarches, lactose, lactose monohydrate, and lactose anhydrous; (3)disintegrating agents such as alginic acid, Primogel, corn starch,lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch,and modified starches, croscarmellose sodium, cross-povidone, sodiumstarch glycolate, and mixtures thereof; (4) lubricants, including agentsthat act on the flowability of a powder to be compressed, includemagnesium stearate, colloidal silicon dioxide, such as Aerosil® 200,talc, stearic acid, calcium stearate, and silica gel; (5) glidants suchas colloidal silicon dioxide; (6) preservatives, such as potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid such as butylparaben, alcohols such asethyl or benzyl alcohol, phenolic compounds such as phenol, orquaternary compounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as Avicel® PH101 and Avicel® PH102;lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose®DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, Magnasweet® (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

E. Kits Comprising the Compositions of the Invention

The present invention also encompasses kits comprising the compositionsof the invention. Such kits can comprise, for example, (1) at least oneGSNOR inhibitor; and (2) at least one pharmaceutically acceptablecarrier, such as a solvent or solution. Additional kit components canoptionally include, for example: (1) any of the pharmaceuticallyacceptable excipients identified herein, such as stabilizers, buffers,etc., (2) at least one container, vial or similar apparatus for holdingand/or mixing the kit components; and (3) delivery apparatus, such as aninhaler, nebulizer, syringe, etc.

F. Methods of Preparing GSNOR Inhibitors

The GSNOR inhibitors of the invention can readily be synthesized usingknown synthetic methodologies or via a modification of known syntheticmethodologies. As would be readily recognized by a skilled artisan, themethodologies described below allow the synthesis of pyrroles having avariety of substituents. Exemplary synthetic methods are described inthe examples below.

According to one synthetic protocol, reaction of 2-furaldehyde with anappropriately substituted acetophenone followed by treatment with astrong acid gives the appropriately substituted 1,4,7-trione.Cyclization of the trione to the corresponding 1,2,5-trisubstitutedpyrrole is readily achieved by reacting the trione with a primary aminein the presence of p-toluenesulfonic acid. In one embodiment of thepresent invention, further derivatization of the phenyl ring at C5 ofthe pyrrole is readily achieved, for example, by various cross-couplingreactions. For example, synthesis of the trisubstituted pyrroles byreacting 1-(4-chlorophenyl)ethanone and 2-furaldehyde will give thetarget pyrrole with 4-chlorophenyl group at C5. The aryl chloride can bederivatized by reaction with a boronic acid under Suzuki couplingconditions. Such routine derivatization methodologies allow the rapidgeneration of compound libraries for in vitro GSNOR inhibition studies.A variety of additional methods are described in Example 1 of thisdocument

If needed, further purification and separation of enantiomers anddiastereomers can be achieved by routine procedures known in the art.Thus, for example, the separation of enantiomers of a compound can beachieved by the use of chiral HPLC and related chromatographictechniques. Diastereomers can be similarly separated. In some instances,however, diastereomers can simply be separated physically, such as, forexample, by controlled precipitation or crystallization.

The process of the invention, when carried out as prescribed herein, canbe conveniently performed at temperatures that are routinely accessiblein the art. In one embodiment, the process is performed at a temperaturein the range of about 25° C. to about 110° C. In another embodiment, thetemperature is in the range of about 40° C. to about 100° C. In yetanother embodiment, the temperature is in the range of about 50° C. toabout 95° C.

Synthetic steps that require a base are carried out using any convenientorganic or inorganic base. Typically, the base is not nucleophilic.Thus, in one embodiment, the base is selected from carbonates,phosphates, hydroxides, alkoxides, salts of disilazanes, and tertiaryamines.

The process of the invention, when performed as described herein, can besubstantially complete after several minutes to after several hoursdepending upon the nature and quantity of reactants and reactiontemperature. The determination of when the reaction is substantiallycomplete can be conveniently evaluated by ordinary techniques known inthe art such as, for example, HPLC, LCMS, TLC, and ¹H NMR.

G. Method of Treatment

The invention encompasses methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through use ofone or more of the disclosed compounds. The methods compriseadministering a therapeutically effective amount of a GSNOR inhibitor toa patient in need. The compositions of the invention can also be usedfor prophylactic therapy.

The GSNOR inhibitor used in the methods of treatment according to theinvention can be: (1) a novel GSNOR inhibitor described herein, or apharmaceutically acceptable salt thereof, a prodrug thereof, or ametabolite thereof; (2) a compound which was known prior to the presentinvention, but wherein it was not known that the compound is a GSNORinhibitor, or a pharmaceutically acceptable salt thereof, a prodrugthereof, or a metabolite thereof; or (3) a compound which was knownprior to the present invention, and wherein it was known that thecompound is a GSNOR inhibitor, but wherein it was not known that thecompound is useful for the methods of treatment described herein, or apharmaceutically acceptable salt thereof, a prodrug thereof, or ametabolite thereof.

The patient can be any animal, domestic, livestock or wild, including,but not limited to cats, dogs, horses, pigs and cattle, and preferablyhuman patients. As used herein, the terms patient and subject may beused interchangeably.

In subjects with deleteriously high levels of GSNOR or GSNOR activity,modulation may be achieved, for example, by administering one or more ofthe disclosed compounds that disrupts or down-regulates GSNOR function,or decreases GSNOR levels. These compounds may be administered withother GSNOR inhibitor agents, such as anti-GSNOR antibodies or antibodyfragments, GSNOR antisense, iRNA, or small molecules, or otherinhibitors, alone or in combination with other agents as described indetail herein.

The present invention provides a method of treating a subject afflictedwith a disorder ameliorated by NO donor therapy. Such a method comprisesadministering to a subject a therapeutically effective amount of a GSNORinhibitor.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition ordisorder. More specifically, “treating” includes reversing, attenuating,alleviating, minimizing, suppressing or halting at least one deleterioussymptom or effect of a disease (disorder) state, disease progression,disease causative agent (e.g., bacteria or viruses), or other abnormalcondition. Treatment is continued as long as symptoms and/or pathologyameliorate.

The disorders can include pulmonary disorders associated with hypoxemiaand/or smooth muscle constriction in the lungs and/or lung infectionand/or lung injury (e.g., pulmonary hypertension, ARDS, asthma,pneumonia, pulmonary fibrosis/interstitial lung diseases, cysticfibrosis, COPD) cardiovascular disease and heart disease, includingconditions such as hypertension, ischemic coronary syndromes,atherosclerosis, heart failure, glaucoma, diseases characterized byangiogenesis (e.g., coronary artery disease), disorders where there isrisk of thrombosis occurring, disorders where there is risk ofrestenosis occurring, chronic inflammatory diseases (e.g., AID dementiaand psoriasis), diseases where there is risk of apoptosis occurring(e.g., heart failure, atherosclerosis, degenerative neurologicdisorders, arthritis and liver injury (ischemic or alcoholic)),impotence, obesity caused by eating in response to craving for food,stroke, reperfusion injury (e.g., traumatic muscle injury in heart orlung or crush injury), and disorders where preconditioning of heart orbrain for NO protection against subsequent ischemic events isbeneficial.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, or a prodrug or metabolitethereof, can be administered in combination with an NO donor. An NOdonor donates nitric oxide or a related redox species and more generallyprovides nitric oxide bioactivity, that is activity which is identifiedwith nitric oxide, e.g., vasorelaxation or stimulation or inhibition ofa receptor protein, e.g., ras protein, adrenergic receptor, NFκB. NOdonors including S-nitroso, O-nitroso, C-nitroso and N-nitroso compoundsand nitro derivatives thereof and metal NO complexes, but not excludingother NO bioactivity generating compounds, useful herein are describedin “Methods in Nitric Oxide Research,” Feelisch et al. eds., pages71-115 (J. S., John Wiley & Sons, New York, 1996), which is incorporatedherein by reference. NO donors which are C-nitroso compounds wherenitroso is attached to a tertiary carbon which are useful herein includethose described in U.S. Pat. No. 6,359,182 and in WO 02/34705. Examplesof S-nitroso compounds, including S-nitrosothiols useful herein,include, for example, S-nitrosoglutathione,S-nitroso-N-acetylpenicillamine, S-nitroso-cysteine and ethyl esterthereof, S-nitroso cysteinyl glycine,S-nitroso-gamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin. Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, isosorbide, nitroglycerin, SIN 1which is molsidomine, furoxamines, N-hydroxy (N-nitrosamine) andperfluorocarbons that have been saturated with NO or a hydrophobic NOdonor.

The combination of a GSNOR inhibitor with R(+) enantiomer of amlodipine,a known NO releaser (Zhang X. P at al. 2002 J. CardiovascularPharmacology 39, 208-214) is also an embodiment of the presentinvention.

The present invention also provides a method of treating a subjectafflicted with pathologically proliferating cells where the methodcomprises administering to said subject a therapeutically effectiveamount of an inhibitor of GSNOR. The inhibitors of GSNOR are thecompounds as defined above, or a pharmaceutically acceptable saltthereof, or a prodrug or metabolite thereof, in combination with apharmaceutically acceptable carrier. Treatment is continued as long assymptoms and/or pathology ameliorate.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating microbes. The microbes involved can bethose where GSNOR is expressed to protect the microbe from nitrosativestress or where a host cell infected with the microbe expresses theenzyme, thereby protecting the microbe from nitrosative stress. The term“pathologically proliferating microbes” is used herein to meanpathologic microorganisms including but not limited to pathologicbacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa,pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata.More detail on the applicable microbes is set forth at columns 11 and 12of U.S. Pat. No. 6,057,367. The term “host cells infected withpathologic microbes” includes not only mammalian cells infected withpathologic viruses but also mammalian cells containing intracellularbacteria or protozoa, e.g., macrophages containing Mycobacteriumtuberculosis, Mycobacterium leper (leprosy), or Salmonella typhi(typhoid fever).

In another embodiment, the pathologically proliferating cells can bepathologic helminths. The term “pathologic helminths” is used herein torefer to pathologic nematodes, pathologic trematodes and pathologiccestodes. More detail on the applicable helminths is set forth at column12 of U.S. Pat. No. 6,057,367.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating mammalian cells. The term “pathologicallyproliferating mammalian cells” as used herein means cells of the mammalthat grow in size or number in said mammal so as to cause a deleteriouseffect in the mammal or its organs. The term includes, for example, thepathologically proliferating or enlarging cells causing restenosis, thepathologically proliferating or enlarging cells causing benign prostatichypertrophy, the pathologically proliferating cells causing myocardialhypertrophy and proliferating cells at inflammatory sites such assynovial cells in arthritis or cells associated with a cellproliferation disorder.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the term “psoriatic condition” refers to disordersinvolving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration. The cell proliferative disordercan be a precancerous condition or cancer. The cancer can be primarycancer or metastatic cancer, or both.

As used herein, the term “cancer” includes solid tumors, such as lung,breast, colon, ovarian, pancreas, prostate, adenocarcinoma, squamouscarcinoma, sarcoma, malignant glioma, leiomyosarcoma, hepatoma, head andneck cancer, malignant melanoma, non-melanoma skin cancers, as well ashematologic tumors and/or malignancies, such as leukemia, childhoodleukemia and lymphomas, multiple myeloma, Hodgkin's disease, lymphomasof lymphocytic and cutaneous origin, acute and chronic leukemia such asacute lymphoblastic, acute myelocytic or chronic myelocytic leukemia,plasma cell neoplasm, lymphoid neoplasm and cancers associated withAIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses andthe like. In one embodiment, proliferative diseases include dysplasiasand disorders of the like.

In one embodiment, the treating cancer comprises a reduction in tumorsize, decrease in tumor number, a delay of tumor growth, decrease inmetastaic lesions in other tissues or organs distant from the primarytumor site, an improvement in the survival of patients, or animprovement in the quality of patient life, or at least two of theabove.

In another embodiment, the treating a cell proliferative disordercomprises a reduction in the rate of cellular proliferation, reductionin the proportion of proliferating cells, a decrease in size of an areaor zone of cellular proliferation, or a decrease in the number orproportion of cells having an abnormal appearance or morphology, or atleast two of the above.

In yet another embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ormetabolite thereof, can be administered in combination with a secondchemotherapeutic agent. In a further embodiment, the secondchemotherapeutic agent is selected from the group consisting oftamoxifen, raloxifene, anastrozole, exemestane, letrozole, cisplatin,carboplatin, paclitaxel, cyclophosphamide, lovastatin, minosine,gemcitabine, araC, 5-fluorouracil, methotrexate, docetaxel, goserelin,vincristin, vinblastin, nocodazole, teniposide, etoposide, epothilone,navelbine, camptothecin, daunonibicin, dactinomycin, mitoxantrone,amsacrine, doxorubicin, epirubicin, idarubicin imatanib, gefitinib,erlotinib, sorafenib, sunitinib malate, trastuzumab, rituximab,cetuximab, and bevacizumab.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ormetabolite thereof, can be administered in combination with an agentthat imposes nitrosative or oxidative stress. Agents for selectivelyimposing nitrosative stress to inhibit proliferation of pathologicallyproliferating cells in combination therapy with GSNOR inhibitors hereinand dosages and routes of administration therefore include thosedisclosed in U.S. Pat. No. 6,057,367, which is incorporated herein.Supplemental agents for imposing oxidative stress (i.e., agents thatincrease GSSG (oxidized glutathione) over GSH (glutathione) ratio orNAD(P) over NAD(P)H ratio or increase thiobarbituric acid derivatives)in combination therapy with GS-FDH inhibitors herein include, forexample, L-buthionine-5-sulfoximine (BSO), glutathione reductaseinhibitors (e.g., BCNU), inhibitors or uncouplers of mitochondrialrespiration and drugs that increase reactive oxygen species (ROS), e.g.,adriamycin, in standard dosages with standard routes of administration.

GSNOR inhibitors may also be co-administered with a phosphodiesteraseinhibitor (e.g., rolipram, cilomilast, roflumilast, Viagra® (sildenifilcitrate), Clalis® (tadalafil), Levitra® (vardenifil), etc.) a β-agonist,a steroid, or a leukotriene antagonist (LTD-4). Those skilled in the artcan readily determine the appropriate therapeutically effective amountdepending on the disorder to be ameliorated.

GSNOR inhibitors may be used as a means to improve β-adrenergicsignaling. In particular, inhibitors of GSNOR alone or in combinationwith β-agonists could be used to treat or protect against heart failure,or other vascular disorders such as hypertension and asthma. GSNORinhibitors can also be used to modulate G protein coupled receptors(GPCRs) by potentiating Gs G-protein, leading to smooth musclerelaxation (e.g., airway and blood vessels), and by attenuating GqG-protein, and thereby preventing smooth muscle contraction (e.g., inairway and blood vessels).

The therapeutically effective amount for the treatment of a subjectafflicted with a disorder ameliorated by NO donor therapy is the GSNORinhibiting amount in vivo that causes amelioration of the disorder beingtreated or protects against a risk associated with the disorder. Forexample, for asthma, a therapeutically effective amount is abronchodilating effective amount; for cystic fibrosis, a therapeuticallyeffective amount is an airway obstruction ameliorating effective amount;for ARDS, a therapeutically effective amount is a hypoxemia amelioratingeffective amount; for heart disease, a therapeutically effective amountis an angina relieving or angiogenesis inducing effective amount; forhypertension, a therapeutically effective amount is a blood pressurereducing effective amount; for ischemic coronary disorders, atherapeutic amount is a blood flow increasing effective amount; foratherosclerosis, a therapeutically effective amount is an endothelialdysfunction reversing effective amount; for glaucoma, a therapeuticamount is an intraocular pressure reducing effective amount; fordiseases characterized by angiogenesis, a therapeutically effectiveamount is an angiogenesis inhibiting effective amount; for disorderswhere there is risk of thrombosis occurring, a therapeutically effectiveamount is a thrombosis preventing effective amount; for disorders wherethere is risk of restenosis occurring, a therapeutically effectiveamount is a restenosis inhibiting effective amount; for chronicinflammatory diseases, a therapeutically effective amount is aninflammation reducing effective amount; for disorders where there isrisk of apoptosis occurring, a therapeutically effective amount is anapoptosis preventing effective amount; for impotence, a therapeuticallyeffective is an erection attaining or sustaining effective amount; forobesity, a therapeutically effective amount is a satiety causingeffective amount; for stroke, a therapeutically effective amount is ablood flow increasing or a TIA protecting effective amount; forreperfusion injury, a therapeutically effective amount is a functionincreasing effective amount; and for preconditioning of heart and brain,a therapeutically effective amount is a cell protective effectiveamount, e.g., as measured by triponin or CPK.

The therapeutically effective amount for the treatment of a subjectafflicted with pathologically proliferating cells means a GSNORinhibiting amount in vivo which is an antiproliferative effectiveamount. Such antiproliferative effective amount as used herein means anamount causing reduction in rate of proliferation of at least about 20%,at least about 10%, at least about 5%, or at least about 1%.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg to 10 g/kg and often ranges from 10 μg to 1 g/kg or 10μg to 100 mg/kg body weight of the subject being treated, per day.

H. Other Uses

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug or metabolite thereof, can be applied tovarious apparatus in circumstances when the presence of such compoundswould be beneficial. Such apparatus can be any device or container, forexample, implantable devices in which a GSNOR inhibitor can be used tocoat a surgical mesh or cardiovascular stent prior to implantation in apatient. The GSNOR inhibitors of the present invention can also beapplied to various apparatus for in vitro assay purposes or forculturing cells.

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug or metabolite thereof, can also be used as anagent for the development, isolation or purification of binding partnersto GSNOR inhibitor compounds, such as antibodies, natural ligands, andthe like. Those skilled in the art can readily determine related usesfor the compounds of the present invention.

EXAMPLES

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

Example 1 General and Specific Methods of Preparing Novel GSNOR PyrroleInhibitors

This example describes schemes for preparing the GSNOR inhibitorsdepicted in Table 1. The synthesis of 6 common intermediates,Intermediates A-F, are first described followed by the syntheticschemes. Some schemes are specific to a particular compound, whileothers are general schemes that include an exemplary method forpreparing a representative compound.

Intermediate Synthesis Intermediate A: Synthesis of ethyl6-(4-bromophenyl)-3,6-dioxohexanoate

Step 1: Synthesis of 4-(4-bromophenyl)-4-oxobutanoic acid (A-1).Anhydrous aluminum trichloride (29.1 g, 218 mmol) was suspended indichloromethane (120 mL) and cooled to 0° C. Bromobenzene (35.1 g, 224mmol) was added carefully. When the addition was complete, succinicanhydride (10.0 g, 100 mmol) was added in ten portions carefully. Thenthe mixture was warmed to room temperature and stirred for 4 h. TLCshowed the reaction was complete, 6N HCl (50 mL) was added dropwise. Thesolid was filtered, washed with distilled water (10 mL×2) and dried invacuo to afford A-1 as a white solid (22 g, yield 82%).

Step 2 and Step 3: Synthesis of ethyl6-(4-bromophenyl)-3,6-dioxohexanoate (Intermediate A). To a solution ofA-1 (5.00 g, 18.6 mmol) in anhydrous MeCN (50 mL) was added CDI (3.91 g,24.2 mmol). The solution was stirred at room temperature for 2 h andturned red to give A-2, which was used for the next step without anypurification. To a suspension of potassium 3-ethoxy-3-oxopropanoate(6.32 g, 37.2 mmol) in anhydrous MeCN (200 mL) TEA (5.63 g, 55.8 mmol)and anhydrous magnesium dichloride (5.3 g, 55.8 mmol) was addedgradually at 0° C. The mixture was stirred at room temperature for 2 h,to which the solution of A-2 was added in portions. The mixture wasstirred at room temperature overnight. The volatiles were removed underreduced pressure and the residue was dissolved in EA (250 mL), washedwith water (50 mL×2) and brine (30 mL), dried over Na₂SO₄, filtered,concentrated and purified by silica gel column chromatography(PE:EA=3:1) to afford Intermediate A as a brown solid (5.3 g, yield88%).

Intermediate B: Synthesis of ethyl6-(4-methoxyphenyl)-3,6-dioxohexanoate

Synthesis of ethyl 6-(4-methoxyphenyl)-3,6-dioxohexanoate.4-(4-methoxyphenyl)-4-oxobutanoic acid (9.5 g, 45.673 mmol) wasconverted to ethyl 6-(4-methoxyphenyl)-3,6-dioxohexanoate following thesame procedure described in step 2 and 3 of the synthesis ofIntermediate A, yielding Intermediate B as a brown solid (10.15 g, 80%).

General Method for Preparing Intermediates C, D, E and F as describedbelow

Representative Procedure: Synthesis of Intermediate C, ethyl7-(4-methoxyphenyl)-4,7-dioxoheptanoate (R2=4-methoxyphenyl)

Step 1: Synthesis of (E)-3-Furan-2-yl-1-(4-methoxy-phenyl)-propenone. Asolution of 2-furaldehyde (5.85 g, 60.92 mmol) was added to a methanolsolution (120 mL) of 4-methoxy acetophenone (8.5 g, 56.6 mmol), followedby the addition of sodium methoxide (3.1 g, 56.6 mmol). The reactionmixture was stirred at room temperature for 16 h, followed by removal ofthe solvent in vacuo. The resultant mixture was diluted with water (130mL) and extracted with ethyl acetate (350 mL). The aqueous layer wasre-extracted with ethyl acetate (100 mL). The combined organic layerswere dried over anhydrous Na₂SO₄ and the solvent was removed in vacuo toobtain the product (E)-3-Furan-2-yl-1-(4-methoxy-phenyl)-propenone as anorange solid (12.6 g, 97%).

Step 2: Synthesis of ethyl 7-(4-methoxyphenyl)-4,7-dioxoheptanoate.Conc. HCl (59 mL) was added to a solution of(E)-3-Furan-2-yl-1-(4-methoxy-phenyl)-propenone (12.6 g, 55.2 mmol) inethanol (237 mL). The reaction mixture was heated under reflux for 16 h,concentrated, and diluted with dichloromethane (250 mL), and theresultant organic layer was washed with water (25 mL). After phaseseparation, the organic layer was dried over anhydrous Na₂SO₄ and thesolvent removed in vacuo to obtain the crude mixture, which was purifiedby silica gel flash chromatography to obtain ethyl7-(4-methoxyphenyl)-4,7-dioxoheptanoate (6.89 g, 43%).

Intermediate D, ethyl 7-(4-bromophenyl)-4,7-dioxoheptanoate(R2=4-bromophenyl)

Followed same procedure described for Intermediate C.

Intermediate E, ethyl 7-(4-bromophenyl)-4,7-dioxoheptanoate(R2=4-bromo-2-methoxyphenyl)

Followed same procedure described for Intermediate C, with the synthesisof the ketone starting material (step 1) described below.

Synthesis of 1-(4-bromo-2-methoxyphenyl)ethanone (Starting material forSynthesis of Intermediate E)

Synthesis of 3-bromophenyl acetate (E-1). To a stirred suspension of3-bromophenol (50 g, 0.29 mol) in pyridine (200 mL) and dichloromethane(100 mL) was added dropwise acetyl chloride (25 mL, 0.35 mol) at 0° C.and the mixture was stirred 18 h at room temperature. LC-MS showed thatthe reaction was complete. Pyridine and dichloromethane was evaporatedin vacuo. Water (600 mL) was added and acidified with hydrochloric acidat pH 2. The reaction mixture was extracted with ethyl acetate (500mL×3) and the organic phase was dried over anhydrous sodium sulfate,filtrated, concentrated and purified by column chromatography(PE:EA=60:1) to afford compound E-1 as a colorless liquid (46 g, 74%).

Synthesis of 1-(4-bromo-2-hydroxyphenyl)ethanone (E-2). To a stirredsuspensions of compound E-1 (46 g, 0.0.21 mol) and anhydrous aluminumchloride power (57 g, 0.42 mol) was heated to 160° C. for 3 h. Themixture reaction was cooled to room temperature and ice (200 g) andwater (800 mL) was poured and purified with hydrochloric acid at pH 7.the reaction was extracted with ethyl acetate (500 mL×3) and the organicphase was washed with saturated sodium bicarbonate, dried over anhydroussodium sulfate, filtrated, concentrated and purified by columnchromatography (PE:EA=60:1) to afford compound E-2 as a light greensolid (35.1 g, 76%).

Synthesis of 1-(4-bromo-2-methoxyphenyl)ethanone (E-3). To a suspensionsof compound E-2 (25 g, 0.12 mol) and potassium carbonate (24 g, 0.18mol) in anhydrous DMF (20 mL) was added to MeI (22.6 mL, 0.23 mol) andthe mixture reaction was stirred at room temperature overnight. LCMSshowed that the reaction was complete. Then water (300 mL) was pouredand the mixture was extracted with ethyl acetate and the organic phasewas (200 mL×3) and the organic phase was washed saturated sodiumchloride, dried over anhydrous sodium sulfate, filtrated, concentratedto afford compound E-3 as a colorless solid (26.1 g, 98%).

Intermediate F, ethyl 7-(4-chloro-2-methoxyphenyl)-4,7-dioxoheptanoate(R2=4-chloro-2-methoxyphenyl)

Followed same procedure described for Intermediate C, with the synthesisof the ketone starting material (step 1) described below.

Synthesis of 1-(4-chloro-2-methoxyphenyl)ethanone (F-3) (Startingmaterial for the Synthesis of Intermediate F)

Synthesis of 1-(4-chloro-2-methoxyphenyl)ethanone (F-3). Followed theprocedure described directly above in the three step synthesis of1-(4-bromo-2-methoxyphenyl)ethanone (E-3).

Representative procedure for Scheme 1: Example where R₂=phenyl. Both4,7-dioxo-7-phenyl-heptanoic acid (an example of 1A) and 1B arecommercial.

Step 1: Preparation of4-[2-(2-carboxy-ethyl)-5-phenyl-pyrrol-1yl]-benzoic acid tert-butylester (1C, R2=phenyl). To a stirred solution of4,7-dioxo-7-phenyl-heptanoic acid (3.57 mmol, 1.0 eq.) in acetic acid(10 ml) was added tert-butyl 4-aminobenzoate (1.05 eq., 3.75 mmol). Themixture was heated to 80° C. for 36 hours. The solvent was evaporated invacuo and the crude residue purified by flash silica-gel chromatography.Gradient elution with ethyl acetate/hexane (0% to 50% ethyl acetate inhexane over 20 minutes) gave the semi-pure product after combination ofthe appropriate fractions and evaporation of the solvent in vacuo. Thetan solid obtained was used without further purification, 59% yield.

Step 2: Preparation of4-[2-(2-carbamoyl-ethyl)-5-phenylpyrrol-1-yl]-benzoic acid tert-butylester (1D, R2=phenyl). To a stirred solution of4-[2-(2-carboxy-ethyl)-5-phenyl-pyrrol-1yl]-benzoic acid tert-butylester (1.33 mmol, 1.0 eq.) in THF (10 ml) was added CDI(1,1′carbonyldiimidazole, 1.60 mmol, 1.2 eq). The mixture was stirred at45° C. for 2 h. To the reaction mixture was then added ammonium acetate(1.72 mmol, 1.3 eq.). The mixture was stirred at 45° C. for 16 hours.

The reaction mixture was diluted with water (100 mL) and extracted withethyl acetate (3×75 mL). The organic phase was washed with 1N citricacid (1×100 mL), water (1×100 mL), and dried (MgSO4). The solvent wasevaporated and the residue was purified by flash silica-gel columnchromatography. Gradient elution with ethyl acetate/hexane (0% to 70%ethyl acetate in hexane over 30 minutes) gave the product as a tan solidafter combination of the appropriate fractions and evaporation of thesolvent in vacuo, 73% yield.

Step 3: Preparation of 4-[2-(2-cyano-ethyl)-5-phenyl-pyrrol-1yl]-benzoicacid tert-butyl ester (1E, R2=phenyl). A stirred solution of4-[2-(2-carbamoyl-ethyl)-5-phenylpyrrol-1-yl]-benzoic acid tert-butylester (3.92 mmol, 1.0 eq.) in dioxane and pyridine was cooled in an icebath. To the mixture was added Trifluoroacetic anhydride (TFA, 7.84mmol, 2.0 eq.). After 10 minutes the ice bath was removed and thereaction was allowed to warm to room temperature overnight. The solventwas evaporated in vacuo and the residue treated with EtOAc (250 mL) andsaturated NaHCO₃ (150 mL). The organic layer was separated, dried(Na₂SO₄) and the solvent evaporated in vacuo. The residue was loadedonto a 40 g RediSep silica-gel column for purification. Gradient elutionwith ethyl acetate/hexane (0% to 10% ethyl acetate in hexane over 25minutes) gave the product as a yellow solid after combination of theappropriate fractions and evaporation of the solvent in vacuo, 62%yield.

Step 4: Preparation of4-{2-phenyl-5-[2-(1H-tetrazol-5-yl)ethyl]-pyrrol-1-yl}-benzoic acidtert-butyl ester (Compound 3 in Table 1, IF where R2=phenyl). To astirred solution of 4-[2-(2-cyano-ethyl)-5-phenyl-pyrrol-1yl]-benzoicacid tert-butyl ester (2.31 mmol, 1.0 eq.) in toluene (12 ml) were addedAzidotrimethylsilane (TMSN₃) (4.62 mmol, 2.0 eq.) and Sn(Bu)₂O(dibutyltin(IV) oxide) (0.23 mmol, 0.1 eq.). The resulting mixture washeated to 110° C. overnight. The solvent was evaporated in vacuo and theresidue loaded onto a 40 g RediSep silica-gel column for purification.Gradient elution with methanol/DCM (0% to 7% methanol in DCM over 25minutes) gave the product4-{2-phenyl-5-[2-(1H-tetrazol-5-yl)ethyl]-pyrrol-1-yl}-benzoic acidtert-butyl ester as a light yellow oil after combination of theappropriate fractions and evaporation of the solvent in vacuo, 62%yield.

Step 5: Preparation of4-{2-phenyl-5-[2-(1H-tetrazol-5-yl)-ethyl]-pyrrol-1-yl}-benzoic acid(Compound 2 in Table 1, 1G where R2=phenyl). A solution of4-{2-phenyl-5-[2-(1h-tetrazol-5-yl)ethyl]-pyrrol-1-yl}-benzoic acidtert-butyl ester (1.34 mmol, 1.0 eq) in formic acid (4.2 ml) was stirredat room temperature for 42 hours. The solvent was evaporated in vacuoand the residue purified by prep HPLC. Removal of the solvent in vacuoyielded 156 mg desired product4-{2-phenyl-5-[2-(1H-tetrazol-5-yl)ethyl]-pyrrol-1-yl}-benzoic acid(32%) and 180 mg byproduct in which the t-butyl group had migrated tothe tetrazole ring (37%).

Step 6: Preparation of4-{2-phenyl-5-[2-(1H-tetrazol-5-yl)-ethyl]-pyrrol-1-yl}-benzamide(Compound 1 in Table 1, IF where R2=phenyl). To a stirred solution of4-{2-phenyl-5-[2-(1h-tetrazol-5-yl)-ethyl]-pyrrol-1-yl}-benzoic acid(0.37 mmol, 1.0 eq.) in DMF(10 ml) were added EDCI (1.48 mmol, 4.0 eq),HOBt hydrate (1.48 mmol, 4.0 eq.), and DIEA (diisopropylethylamine, 2.96mmol, 8.0 eq.). The resulting mixture was stirred at room temperatureovernight. The reaction mixture was diluted with DCM (75 mL) and 10%citric acid (50 mL). The organic layer was washed with saturated NaHCO₃(75 mL). The saturated NaHCO₃ was acidified with 1M HCl and extractedwith DCM (2×100 mL). The organic layer was dried (MgSO₄) and the solventremoved in vacuo. The crude material was purified by prep HPLC to giveproduct as a white solid, 30% yield.

Representative procedure for Scheme 2: Synthesis of4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzamide(2H, R2=4-methoxyphenyl)

Step 1: Preparation of3-(1-(4-(tert-butoxycarbonyl)-2-methylphenyl)-5-(4-methoxyphenyl)-1H-pyrrol-2-yl)propanoicacid (2C, R2=4-methoxyphenyl). (550 mg, 2.083 mmol) andtert-butyl-4-amino-3-methylbenzoate (431 mg, 2.083 mmol) was dissolvedin anhydrous dioxane (5 mL). TsOH (35 mg, 0.208 mmol) was added and thereaction mixture was heated to 100° C. for 8 hours. TLC and LC-MS showedno starting material. The mixture was diluted with water (10 mL) and EA(10 mL). The organic layer was separated and the aqueous phase wasextracted with EA (10 mL×3). The combined organic layers were dried withmagnesium sulfate, filtered, concentrated and purified by silica gelcolumn chromatography to afford compound 2C, R2=4-methoxyphenyl as abrown oil (556 mg, 61%).

Step 2: Preparation of tert-butyl4-(2-(3-amino-3-oxopropyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(2D, R2=4-methoxyphenyl).3-(1-(4-(tert-butoxycarbonyl)-2-methylphenyl)-5-(4-methoxyphenyl)-1H-pyrrol-2-yl)propanoicacid (500 mg, 1.15 mmol) was dissolved in anhydrous THF (10 mL). CDI(230 mg, 1.38 mmol) was added in five portions and stirred at roomtemperature for 2 h. TLC showed no starting material. The yellowsolution was added dropwise to 25% ammonium hydrate (10 mL) at 0° C.After the addition was complete, the mixture was allowed to warm to roomtemperature and stirred for 2 h. The solution was extracted with ethylacetate (10 mL×3) and the combined organic layers were washed with 5%hydrochloric acid, saturated sodium bicarbonate and brine separately,dried over MgSO₄, filtered and concentrated to obtain the title compound2D, R2=4-methoxyphenyl as a yellow solid (320 mg, 65%).

Step 3: Preparation of tert-butyl4-(2-(2-cyanoethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(2E, R2=4-methoxyphenyl). Tert-butyl4-(2-(3-amino-3-oxopropyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(355 mg, 0.818 mmol) was dissolved in anhydrous THF (2 mL).Triethylamine (206 mg, 2.05 mmol) was added and the mixture was cooledto 0° C. TFAA was added dropwise and stirred at this temperature for 30min. TLC showed that the reaction was complete. Diluted with Ethylacetate (10 mL) and washed with brine, the organic phase was separatedand dried with magnesium sulfate, filtered, concentrated and purified bycolumn chromatography to afford compound 2E, R2=4-methoxyphenyl as abrown oil (280 mg, 82%).

Step 4: Preparation of tert-butyl4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(2F, R2=4-methoxyphenyl). Tert-butyl4-(2-(2-cyanoethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(280 mg, 0.673 mmol) was dissolved in toluene (3 mL). Bu₂SnO (30 mg,0.135 mmol) and TMSN₃ (1.32 g, 12.1 mmol) were added and the mixture washeated to reflux overnight. Toluene was evaporated in vacuo and theresidue was purified by silica gel column chromatography to afford 2F,R2=4-methoxyphenyl as a yellow oil (265 mg, 86%).

Step 5: Preparation of4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoicacid (2G, R2=4-methoxyphenyl). Tert-butyl4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(265 mg) was dissolved in formic acid (3 mL) and stirred at roomtemperature overnight. LC-MS showed that the reaction was complete.Formic acid was evaporated and the residue was purified by prep-TLC toafford 2G, R2=4-methoxyphenyl as a yellow solid (130 mg, 56%).

Step 6: Preparation of4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzamide(2H, R2=4-methoxyphenyl).4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzoicacid (130 mg, 0.332 mmol) was dissolved in anhydrous THF (3 mL). CDI (68mg, 0.419 mmol) was added in three portions at room temperature andstirred at this temperature for 1 h. The resultant yellow solution wasadded dropwise to 25% ammonium hydrate (3 mL) at 0° C. After theaddition was complete, the mixture was allowed to warm to roomtemperature and stirred for 2 h. The mixture was evaporated in vacuo andthe residue was purified by prep-HPLC to afford 2H, R2=4-methoxyphenylas a yellow solid (7 mg, 5.4%).

Representative procedure for Scheme 3: Synthesis of4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol(3F, R2=4-bromophenyl)

Step 1: Preparation of ethyl3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanoate (3B,R2=4-bromophenyl). Ethyl 7-(4-bromophenyl)-4,7-dioxoheptanoate (3A whereR2=4-bromophenyl) (4.0 g, 11.76 mmol) was dissolved in anhydrousdioxane. 4-Aminophenol (1.30 g, 11.76 mmol) and TsOH (203 mg, 1.18 mmol)was added and the mixture was heated to 110° C. overnight. TLC and LC-MSshowed that the reaction was complete. Dioxane was evaporated in vacuoand the residue was purified by silica gel column chromatography toafford 3B, R2=4-bromophenyl as a brown solid (2.90 g, 60%).

Step 2: Preparation of3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanoic acid(3C, R2=4-bromophenyl). Ethyl3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanoate (1.0g, 2.42 mmol) was dissolved in THF/H₂O (10 mL, v/v=1:1), lithiumhydroxide monohydrate (254 mg, 6.05 mmol) was added and the mixture wasstirred at room temperature overnight. TLC showed the starting materialwas consumed. THF was evaporated in vacuo and the residue was acidifiedto pH=4 with 5% hydrochloric acid. White precipitate was filtered anddried in vacuo to afford 3C, R2=4-bromophenyl as a yellow solid (900 mg,97%).

Step 3: Preparation of3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanamide (3D,R2=4-bromophenyl).3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanoic acidwas converted to 3D, R2=4-bromophenyl following the same proceduredescribed in Scheme 2, step 2 to give product as a yellow solid (96%yield).

Step 4: Preparation of3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanenitrile(3E, R2=4-bromophenyl).3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanamide wasconverted to 3E, R2=4-bromophenyl following the same procedure describedin Scheme 2, step 3 to give product as a brown oil (160 mg, 38%).

Step 5: Preparation of4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol(3F, R2=4-bromophenyl).3-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)propanenitrilewas converted to 3F, R2=4-bromophenyl following the same proceduredescribed in Scheme 2, step 4, where purification was accomplished byprep-HPLC to afford desired product as a yellow solid (75 mg, 48%).

Representative procedure for Scheme 4: Synthesis of4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol(4F, R2=4-bromophenyl)

Step 1: Preparation of ethyl2-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)acetate (4B,R2=4-bromophenyl). Ethyl 6-(4-bromophenyl)-3,6-dioxohexanoate(Intermediate B) (5 g, 15.0 mmol) was converted to 4B, R2=4-bromophenylfollowing the same procedure described in Scheme 3, step 1 withpurification by silica gel column chromatography (PE:EA=5:1) to afford4B, R2=4-bromophenyl as a yellow solid (3.2 g, yield 52%).

Step 2: Preparation of2-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl) acetic acid(4C, R2=4-bromophenyl). To a solution of ethyl24544-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)acetate (2.0 g,5.013 mmol) in THF/H₂O (v/v=1/1, 20 mL) was added LiOH H₂O (632 mg,15.038 mmol). The solution was stirred at room temperature for 3 h. THFwas evaporated in vacuo and the resultant aqueous solution was acidifiedwith 10% HCl to pH=3.0. The resultant precipitate was isolated byfiltration, rinsed with water (50 mL) and dried in vacuo to afford 4C,R2=4-bromophenyl (1.380 g, yield 74%) as a brown powder.

Step 3: Preparation of2-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)acetamide (4D,R2=4-bromophenyl). To a solution of2-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)acetic acid (1.3g, 3.504 mmol) in anhydrous THF (30 mL) was added CDI (0.851 g, 5.256mmol) in portions. Stirred at room temperature for 2 h. Then it wasadded to con. NH₃ (80 mL) and stirred at room temperature for 2 h. Thevolatiles were evaporated in vacuo and the residue was extracted withethyl acetate (50 mL) twice. The combined organic layers were washedwith water (30 mL×2) and brine (20 mL), dried over Na₂SO₄, filtered andconcentrated to afford 4D, R2=4-bromophenyl as brown oil (1.45 g, yield100%).

Step 4: Preparation of2-(5-(4-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)acetonitrile(4E, R2=4-bromophenyl). To a solution of24544-bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)acetamide (1.45 g,3.919 mmol) in THF (30 mL) was added Et₃N (3.958 g, 39.189 mmol)dropwise, then TFAA (3 mL) was added dropwise at 0-10° C. The mixturewas stirred at room temperature for 0.5 h. The volatiles were removedunder reduced pressure and the residue was extracted with ethyl acetate(50 mL×2), washed with water (30 mL) and brine (20 mL), dried overNa₂SO₄, concentrated and purified by silica gel column chromatography(PE:EA=4:1) to afford 4E, R2=4-bromophenyl as a yellow solid (1.2 g,yield 87%).

Step 5: Preparation4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol(4F, R2=4-bromophenyl).2-(5-(4-Bromophenyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)acetonitrile(4E, R2=4-bromophenyl) (430 mg, 1.141 mmol) was converted to 4F,R2=4-bromophenyl following the same procedure described in Scheme 2,step 4 with purification by silica gel column chromatography (PE:EA=1:2)to afford 4F, R2=4-bromophenyl as yellow powder (393 mg, yield 82%).

Representative procedure for Scheme 5: Synthesis of4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzamide(5H, R2=4-bromophenyl)

Step 1: Preparation of tert-butyl4-(2-(4-bromophenyl)-5-(2-ethoxy-2-oxoethyl)-1H-pyrrol-1-yl)-3-methylbenzoate(5B, R2=4-bromophenyl). Ethyl 6-(4-bromophenyl)-3,6-dioxohexanoate (5.0g, 15.0 mmol), tert-butyl-4-amino-3-methylbenzoate (3.105 g, 15.0 mmol)and 4-toluenesulfonic acid (258 mg, 1.500 mmol) in anhydrous dioxane wasrefluxed for 12 h. The solvent evaporated in vacuo and the residue wasextracted with ethyl acetate (200 mL×2), washed with water (100 mL) andbrine (50 mL), dried over Na₂SO₄, concentrated and purified by silicagel column chromatography (PE:EA=4:1) to afford 5B, R2=4-bromophenyl asbrown oil (4.1 g, yield 55%).

Step 2: Preparation of2-(5-(4-bromophenyl)-1-(4-(tert-butoxycarbonyl)-2-methylphenyl)-1H-pyrrol-2-yl)aceticacid (5C, R2=4-bromophenyl). tert-Butyl4-(2-(4-bromophenyl)-5-(2-ethoxy-2-oxoethyl)-1H-pyrrol-1-yl)-3-methylbenzoate(2.0 g, 4.024 mmol) was converted to 5C, R2=4-bromophenyl following thesame procedure described in Scheme 3, step 2 to give 5C,R2=4-bromophenyl (1.30 mg, yield 69%) as a brown powder.

Step 3: Preparation of tert-butyl4-(2-(2-amino-2-oxoethyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(5D, R2=4-bromophenyl). To a solution of2-(5-(4-bromophenyl)-1-(4-(tert-butoxycarbonyl)-2-methylphenyl)-1H-pyrrol-2-yl)aceticacid (1.30 g, 2.772 mmol) in anhydrous THF (30 mL) was added CDI (0.525g, 3.603 mmol) in portions. The mixture was stirred at room temperaturefor 2 h. Then it was added to con.NH₃.H₂O (60 mL), stirred at roomtemperature for 2 h. The volatiles were removed under reduced pressureand the residue was extracted with ethyl acetate (50 mL×2), washed withwater (30 mL) and brine (20 mL), dried over Na₂SO₄, concentrated toafford 5D, R2=4-bromophenyl as a brown oil (1.30 g, yield 100%).

Step 4: Preparation of tert-butyl4-(2-(4-bromophenyl)-5-(cyanomethyl)-1H-pyrrol-1-yl)-3-methylbenzoate(5E, R2=4-bromophenyl). To a solution of tert-butyl4-(2-(2-amino-2-oxoethyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(1.30 g, 2.776 mmol) in THF (20 mL) was added Et₃N (2.805 g, 27.778mmol) dropwise, then TFAA (2.5 mL) was added dropwise at 0-10° C. Themixture was stirred at room temperature for 0.5 h. The volatiles wereremoved under reduced pressure and the residue was extracted with ethylacetate (50 mL×2), washed with water (30 mL) and brine (20 mL), driedover Na₂SO₄, concentrated and purified by silica gel columnchromatography (PE:EA=5:1) to afford 5E, R2=4-bromophenyl as a yellowsolid (730 mg, yield 58%).

Step 5: Preparation of tert-butyl4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(5F, R2=4-bromophenyl). To a solution of tert-butyl4-(2-(4-bromophenyl)-5-(cyanomethyl)-1H-pyrrol-1-yl)-3-methylbenzoate(730 mg, 1.662 mmol) in Toluene (20 mL), was added TMSN₃ (3.731 g,32.444 mmol) in portions. The mixture was stirred at 110° C. overnight.The solvent was removed under reduced pressure and the residue wasextracted with ethyl acetate (50 mL×2), washed with water and brine,dried over Na₂SO₄, concentrated and purified by silica gel columnchromatography (PE:EA=1:2) to afford 5F, R2=4-bromophenyl as yellowpowder (520 mg, yield 80%).

Step 6: Preparation of4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzoicacid (5G, R2=4-bromophenyl). The solution of tert-butyl4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzoate(730 mg, 1.662 mmol) in formic acid (10 mL) was stirred at roomtemperature for 24 h. The solvent was removed with toluene under reducedpressure to afford 5G, R2=4-bromophenyl as yellow powder (500 mg, yield100%).

Step 7: Preparation of4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzamide(5H, R2=4-bromophenyl). To a solution of4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzoicacid (200 mg, 0.458 mmol) in anhydrous DMF (3 mL) was added HOBt (74 mg,0.549 mmol) and EDCI (132 mg, 0.686 mmol). The mixture was stirred at20° C. and checked by TLC until the material disappeared. Then DIPEA(158 mg, 1.143 mmol) and NH₄Cl (122 mg, 2.286 mmol) was added, stirredovernight. The crude product was purified by Prep HPLC to afford 5H,R2=4-bromophenyl as white powder (68 mg, yield 34%)

Representative procedure for Scheme 6: Synthesis of4-(2-(4-(1H-imidazol-1-yl)phenyl)-5-(2-(1H-tetrazol-5-yl)ethyl)-1H-pyrrol-1-yl)phenol(6B, R1=4-hydroxyphenyl).4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol(40 mg, 0.0978 mmol) and imidazole (10 mg, 0.1467 mmol) was dissolved inanhydrous DMSO (0.4 mL), CuI (6 mg, 0.0293 mmol), L-proline (6 mg,0.0479 mmol) and potassium carbonate (27 mg, 0.1956 mmol) was added andpurged with nitrogen for 5 min. Then the mixture was heated to 110° C.for 1 h by microwave irradiation. The reaction mixture was diluted withTHF (15 mL) and filtered. The filtrate was concentrated and purified byprep-HPLC to afford 6B, R1=4-hydroxyphenyl (5 mg, yield 12%).

Representative procedure for Scheme 7: Synthesis of4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-1-yl)phenol.To a mixture of4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol(1.0 g, 2.52 mmol) and 2-methyl-1H-imidazole (2.07 g, 25.2 mmol) in DMSO(10.0 mL) was added 8-HQ (54 mg, 0.38 mmol), Cu₂O (0.29 g, 2.52 mmol),Cs₂CO₃ (0.986 g, 3.03 mmol) and PEG-2000 (0.29 g). The resultant mixturewas heated at 150° C. for 3 h under microwave irradiation. The mixturewas treated with EA (60 mL), the resultant precipitate was filtered andpurified by Biotage-flash (Solid phase: C18; Mobile phase: A: CH₃CN, B:H₂O (0.05% CF₃COOH); gradient: 12%→24% B in A; flow rate: 40 mL/min) toafford an off white solid (143 mg, yield 14%)

Representative procedure for Scheme 8: Synthesis of4-(2-((1H-tetrazol-5-yl)methyl)-5-(5-(2-methyl-1H-imidazol-1-yl)thiophen-2-yl)-1H-pyrrol-1-yl)-3-methylbenzamide(8E, R1=4-carbamoyl-2-methylphenyl)

Step 1: Synthesis of 6-(5-bromothiophen-2-yl)-3,6-dioxohexanenitrile(8B). To a solution of 2-cyanoacetic acid (13 g, 50 mmol) in THF (100mL) was added dropwise i-PrMgCl (16 g, 250 mmol) at −78° C. for 1 h. Asolution of 4-(5-bromothiophen-2-yl)-4-oxobutanoic acid (8A) and CDI (12g, 74 mmol) in THF was added dropwise at −78° C. and the resultantmixture was stirred at −78° C. for 1 h and at room temperature for 1 h.The reaction mixture was quenched with saturated ammonium chloride (200mL). The organic phase was separated, dried over Na₂SO₄, filtered,concentrated and purified by silica gel column chromatography(PE:EA=5:1) to afford 8B as a black oil (9.7 g, 69%).

Step 2: Synthesis of4-(2-(5-bromothiophen-2-yl)-5-(cyanomethyl)-1H-pyrrol-1-yl)-3-methylbenzamide(8C, R1=4-carbamoyl-2-methylphenyl). To a solution of 8B (2.85 g, 10mmol) in EtOH was added 4-amino-3-methylbenzamide (1.80 g, 12 mmol) andTsOH (190 mg, 1.0 mmol). The reaction mixture was stirred at 90° C.overnight. TLC and LC-MS showed that the reaction was complete. EtOH wasevaporated in vacuo and the residue was purified by silica gel columnchromatography (PE:EA=2:1) to afford 8C, R1=4-carbamoyl-2-methylphenylas a red solid (2.60 g, 65%).

Step 3: Synthesis of4-(2-((1H-tetrazol-5-yl)methyl)-5-(5-bromothiophen-2-yl)-1H-pyrrol-1-yl)-3-methylbenzamide(8D, R1=4-carbamoyl-2-methylphenyl).4-(2-(5-bromothiophen-2-yl)-5-(cyanomethyl)-1H-pyrrol-1-yl)-3-methylbenzamide(8-3, R1=4-carbamoyl-2-methylphenyl) was converted to the title compound(8D, R1=4-carbamoyl-2-methylphenyl) following the same proceduredescribed in Scheme 5, step 5.

Step 4: Synthesis of4-(2-((1H-tetrazol-5-yl)methyl)-5-(5-(2-methyl-1H-imidazol-1-yl)thiophen-2-yl)-1H-pyrrol-1-yl)-3-methylbenzamide(8E, R1=4-carbamoyl-2-methylphenyl).4-(2-((1H-tetrazol-5-yl)methyl)-5-(5-bromothiophen-2-yl)-1H-pyrrol-1-yl)-3-methylbenzamidewas converted to the title compound (8E, R1=4-carbamoyl-2-methylphenyl)following the coupling procedure described in Scheme 7.

Representative procedure for Scheme 9: Synthesis of4-(1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide(9K, R=2-(1H-tetrazol-5-yl) ethyl)

Synthesis of 9B, (methyl 4-bromo-2-methylbenzoate). To a solution ofcompound 9A (79.45 g, 369.5 mmol) in methanol (450 mL) was added thionylchloride (53.6 mL, 739 mmol), and the mixture was stirred at 70° C. for6 h. The reaction mixture was concentrated to afford 9B (86.37 g, yield100%).

Synthesis of 9C, (methyl 4-cyano-2-methylbenzoate). A mixture of 9B(methyl 4-bromo-2-methylbenzoate) (84.65 g, 369.5 mmol) and cuprouscyanide (45 g, 502.5 mmol) in dimethylformamide (60 mL) was refluxed for8 h. After being cooled down to room temperature, the reaction mixturewas diluted with water (400 mL), and the resulting mixture wasfiltrated. The filter cake was washed with ethyl acetate (200 mL×3). Thefiltrate was washed with water (400 mL), and the aqueous layer wasextracted with ethyl acetate (200 mL×2). The combined organic layer waswashed with brine (400 mL), dried over anhydrous sodium sulfate (40 g),and evaporated. The residue was purified by silica gel columnchromatography (petroleum ether/ethyl acetate=15:1) to afford 9C,(methyl 4-cyano-2-methylbenzoate) (38 g, yield 58.7%) as a yellow solid.

Synthesis of 9D, (4-cyano-2-methylbenzoic acid). To a solution of 9C(methyl 4-cyano-2-methylbenzoate) (38 g, 0.217 mol) in ethanol (450 mL)was added aqueous sodium hydroxide solution (150 mL, 2.9 M/L), and themixture was stirred at room temperature for 12 h. Most of ethanol wasremoved under reduced pressure, and the residue was diluted with water(300 mL). The aqueous layer was washed with ethyl ether (200 mL×2), andthe aqueous layer was acidified with hydrochloric acid (450 mL, 1 M/L)under 0° C. to PH<6. The mixture was extracted with ethyl acetate (400mL×3), and the organic layer was washed with brine, dried, andevaporated to give 9D (4-cyano-2-methylbenzoic acid) (40 g, yield 100%)as a yellow solid.

Synthesis of 9E, (4-cyano-2-methylbenzoyl chloride). To a solution of 9D(4-cyano-2-methylbenzoic acid) (20 g, 0.124 mol) in toluene (200 mL) wasadded thionyl chloride (18 mL, 0.248 mol), and catalytic amount ofdimethylformamide (0.5 mL). The resultant mixture was stirred at 70° C.for 14 h, and concentrated under reduced pressure to give 9E(4-cyano-2-methylbenzoyl chloride) (24 g, yield 100%), which was used inthe next step without further purification.

Synthesis of 9F, (ethyl3-(4-cyano-2-methylphenyl)-2-(4-methoxyphenyl)-3-oxopropanoate). To asolution of (4-methoxy-phenyl)-acetic acid ethyl ester (25.96 g, 133.7mmol) in anhydrous tetrahydrofuran (200 mL) was added lithiumhexamethyldisilazide (200 mL, 1 mol/L) at −78° C. under nitrogen. Afterbeing stirred for 15 min., a solution of 9E (4-cyano-2-methylbenzoylchloride) (24 g, 133.6 mmol) in anhydrous tetrahydrofuran (150 mL) wasadded dropwise, and the resulting mixture was stirred at roomtemperature overnight for 16 h. The reaction mixture was quenched by theaddition of saturated aqueous ammonium chloride solution (200 mL), andthe resultant mixture was extracted with ethyl acetate (100 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate (20 g),filtered, and evaporated. The residue was purified on silica gel column(petroleum ether/ethyl acetate=25:1) to give 9F (ethyl3-(4-cyano-2-methylphenyl)-2-(4-methoxyphenyl)-3-oxopropanoate) (40 g,yield 88.7%).

Synthesis of 9G, (4-(2-(4-methoxyphenyl)acetyl)-3-methylbenzonitrile).To a mixture of ethyl3-(4-cyano-2-methylphenyl)-2-(4-methoxyphenyl)-3-oxopropanoate (9F) (40g, 0.1186 mol) in dimethyl sulfoxide (75 mL) was added catalytic amountof brine (2.5 mL), and the reaction mixture was heated at 150° C. for2.5 h. When TLC showed the starting material was consumed, the reactionmixture was cooled down to room temperature, and partitioned betweenwater (150 mL) and ethyl acetate (150 mL). The aqueous layer wasextracted with ethyl acetate (100 mL×2). The combined organic layer waswashed with brine (200 mL), dried over anhydrous sodium sulfate (15 g),evaporated, and purified by chromatography on silica gel (petroleumether/ethyl acetate=25:1) to give4-(2-(4-methoxyphenyl)acetyl)-3-methylbenzonitrile (9G) (19 g, yield60.4%).

Synthesis of 9H,(4-(2-(4-methoxyphenyl)pent-4-enoyl)-3-methylbenzonitrile). To asolution of 9G (4-(2-(4-methoxyphenyl)acetyl)-3-methylbenzonitrile) (19g, 71.6 mmol) in anhydrous tetrahydrofuran (200 mL) was added dropwiselithium hexamethyldisilazide (LiHMDS) (86 mL, 1 mol/L in THF) at −78° C.under nitrogen, and the reaction mixture was stirred at −78° C. for 30min. A solution of 3-bromo-propene (10.4 g, 86 mmol) in anhydroustetrahydrofuran (70 mL) was added dropwise into the mixture, and theresulting mixture was warmed to room temperature and stirred overnight.The reaction was quenched by the addition of saturated aqueous ammoniumchloride solution (200 mL), and extracted with ethyl acetate (100 mL×3).The combined organic layers were washed with brine (200 mL), dried overanhydrous sodium sulfate (10 g), evaporated, and purified on silica gelcolumn (petroleum ether/ethyl acetate=25:1) to give 9H(4-(2-(4-methoxyphenyl)pent-4-enoyl)-3-methylbenzonitrile) (7.7 g, yield35.2%) as an oil.

Synthesis of 9I,(4-(2-(4-methoxyphenyl)-4-oxobutanoyl)-3-methylbenzonitrile). To asolution of 9H(4-(2-(4-methoxyphenyl)pent-4-enoyl)-3-methylbenzonitrile) (9.1 g, 29.8mmol) in dichloromethane (400 mL) at −78° C. was bubbled with ozone in40 min. Dimethyl sulfide (20 mL) was slowly added into the solution at−78° C. After the addition, the resulting mixture was stirred at roomtemperature overnight, poured into water (150 mL), and extracted withdichloromethane (150 mL×2). The organic layer was washed with brine (150mL), dried over anhydrous sodium sulfate (15 g), evaporated, andpurified by silica gel (petroleum ether/ethyl acetate=5:1) to give 9I,4-(2-(4-methoxyphenyl)-4-oxobutanoyl)-3-methylbenzonitrile (3.0 g, yield32.8%) as a yellow solid.

Synthesis of4-(1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzonitrile,(9J, R=2-(1H-tetrazol-5-yl) ethyl). A mixture of4-(2-(4-methoxyphenyl)-4-oxobutanoyl)-3-methylbenzonitrile (9I) (200 mg,0.65 mmol), 2-(1H-tetrazol-5-yl)ethanamine hydrochloride (146 mg, 0.98mmol) in a mixture of HOAc (78 mg, 1.3 mmol) in EtOH (10 mL) was stirredat 120° C. in microwave for 0.5 hour. When LCMS showed desired productwas formed, the mixture was concentrated in vacuo to afford the crudecompound 9J, R=2-(1H-tetrazol-5-yl) ethyl (250 mg), which was useddirectly in the next step.

Synthesis of4-(1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide,(9K, R=2-(1H-tetrazol-5-yl) ethyl). To a solution of4-(1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzonitrile(9J, R=2-(1H-tetrazol-5-yl) ethyl) (250 mg, 0.65 mmol) in DMSO (5 mL)was added a solution of NaOH (52 mg, 1.3 mmol) in a mixture of water(0.5 mL) and H₂O₂ (74 mg, 0.65 mmol). After being stirred at roomtemperature for 4 hours, the mixture was purified by preparative HPLC togive 24 mg of4-(1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide(9K, R=2-(1H-tetrazol-5-yl) ethyl) (yield 9% in two steps) as a greysolid.

Example 2 GSNOR Assays

Various compounds were tested in vitro for their ability to inhibitGSNOR activity. Representative compounds and their corresponding GSNORactivity are described in a paragraph before Table 1 above. GSNORexpression and purification is described in Biochemistry 2000, 39,10720-10729.

GSNOR fermentation: Pre-cultures were grown from stabs of a GSNORglycerol stock in 2XYT media containing 100 ug/ml ampicillin after anovernight incubation at 37° C. Cells were then added to fresh 2XYT (4L)containing ampicillin and grown to an OD (A₆₀₀) of 0.6-0.9 at 37° C.before induction. GSNOR expression was induced with 0.1% arabinose in anovernight incubation at 20° C.

GSNOR Purification: E. coli cell paste was lysed by nitrogen cavitationand the clarified lysate purified by Ni affinity chromatography on anAKTA FPLC (Amersham Pharmacia). The column was eluted in 20 mM Tris pH8.0/250 mM NaCl with a 0-500 mM imidazole gradient. Eluted GSNORfractions containing the Smt-GSNOR fusion were digested overnight withUlp-1 at 4° C. to remove the affinity tag then re-run on the Ni columnunder the same conditions. GSNOR was recovered in the flowthroughfraction and for crystallography is further purified by Q-Sepharose andHeparin flowthrough chromatography in 20 mM Tris pH 8.0, 1 mM DTT, 10 uMZnSO₄.

GSNOR assay: GSNO and Enzyme/NADH Solutions are made up fresh each day.The Solutions are filtered and allowed to warm to room temperature. GSNOSolution: 100 mM NaPO4 (pH 7.4), 0.480 mM GSNO. 396 μL of GSNO Solutionis added to a cuvette followed by 8 μL of test compound in DMSO (or DMSOonly for full reaction control) and mixed with the pipette tip.Compounds to be tested are made up at a stock concentration of 10 mM in100% DMSO. 2 fold serial dilutions are done in 100% DMSO. 8 μL of eachdilution are added to an assay so that the final concentration of DMSOin the assay is 1%. The concentrations of compounds tested range from100 to 0.003 μM. Enzyme/NADH Solution: 100 mM NaPO4 (pH 7.4), 0.600 mMNADH, 1.0 μg/mL GSNO Reductase. 396 μL of the Enzyme/NADH Solution isadded to the cuvette to start the reaction. The cuvette is placed in theCary 3E UV/Visible Spectrophotometer and the change in 340 nmabsorbance/min at 25° C. is recorded for 3 minutes. The assays are donein triplicate for each compound concentration. IC50's for each compoundare calculated using the standard curve analysis in the Enzyme KineticsModule of SigmaPlot.

Final assay conditions: 100 mM NaPO4, pH 7.4, 0.240 mM GSNO, 0.300 mMNADH, 0.5 μg/mL GSNO Reductase and 1% DMSO. Final volume: 800μL/cuvette.

Example 3 Efficacy of GSNORi in experimental asthma

Experimental Asthma Model:

A mouse model of ovalbumin (OVA)-induced asthma is used to screen GSNORinhibitors for efficacy against methacholine (MCh)-inducedbronchoconstriction/airway hyper-reactivity. This is a widely used andwell characterized model that presents with an acute, allergic asthmaphenotype with similarities to human asthma. Efficacy of GSNORinhibitors are assessed using a prophylactic protocol in which GSNORinhibitors are administered prior to challenge with MCh.Bronchoconstriction in response to challenge with increasing doses ofMCh is assessed using whole body plethysmography (P_(enh); Buxco). Theamount of eosinophil infiltrate into the bronchoaveolar lavage fluid(BALF) is also determined as a measure of lung inflammation. The effectof GSNOR inhibitors are compared to vehicles and to Combivent (inhaled;IH) as the positive control.

Materials and Methods

Allergen Sensitization and Challenge Protocol

OVA (500 μg/ml) in PBS is mixed with equal volumes of 10% (w/v) aluminumpotassium sulfate in distilled water and incubated for 60 min. at roomtemperature after adjustment to pH 6.5 using 10 N NaOH. Aftercentrifugation at 750×g for 5 min, the OVA/alum pellet is resuspended tothe original volume in distilled water. Mice receive an intraperitoneal(IP) injection of 100 μg OVA (0.2 mL of 500 μg/mL in normal saline)complexed with alum on day 0. Mice are anesthetized by IP injection of a0.2-mL mixture of ketamine and xylazine (0.44 and 6.3 mg/mL,respectively) in normal saline and are placed on a board in the supineposition. Two hundred fifty micrograms (100 μl of a 2.5 mg/ml) of OVA(on day 8) and 125 μg (50 μl of 2.5 mg/ml) OVA (on days 15, 18, and 21)are placed on the back of the tongue of each animal.

Pulmonary Function Testing (Penh)

In vivo airway responsiveness to methacholine is measured 24 h after thelast OVA challenge in conscious, freely moving, spontaneously breathingmice with whole body plethysmography using a Buxco chamber (Wilmington,N.C.). Mice are challenged with aerosolized saline or increasing dosesof methacholine (5, 20 and 50 mg/mL) generated by an ultrasonicnebulizer for 2 min. The degree of bronchoconstriction is expressed asenhanced pause (P_(enh)), a calculated dimensionless value, whichcorrelates with the measurement of airway resistance, impedance, andintrapleural pressure in the same mouse. P_(enh) readings are taken andaveraged for 4 min. after each nebulization challenge. P_(enh) iscalculated as follows: P_(enh)=[(T_(e)/T_(r)−1)×(PEF/PIF)], where T_(e)is expiration time, T_(r) is relaxation time, PEF is peak expiratoryflow, and PIF is peak inspiratory flow×0.67 coefficient. The time forthe box pressure to change from a maximum to a user-defined percentageof the maximum represents the relaxation time. The T_(r) measurementbegins at the maximum box pressure and ends at 40%.

Eosinophil Infiltrate in BALF

After measurement of airway hyper-reactivity, the mice are exsanguinatedby cardiac puncture, and then BALF is collected from either both lungsor from the right lung after tying off the left lung at the mainstembronchus. Total BALF cells are counted from a 0.05 mL aliquot, and theremaining fluid is centrifuged at 200×g for 10 min at 4° C. Cell pelletsare resuspended in saline containing 10% BSA with smears made on glassslides. Eosinophils are stained for 5 min. with 0.05% aqueous eosin and5% acetone in distilled water, rinsed with distilled water, andcounterstained with 0.07% methylene blue.

GSNOR Inhibitors and Controls

GSNOR inhibitors are reconstituted in phosphate buffered saline (PBS),pH 7.4, at concentrations ranging from 0.00005 to 3 mg/mL. GSNORinhibitors are administered to mice (10 mL/kg) as a single dose eitherintravenously (IV) or orally via gavage. Dosing is performed from 30min. to 24 h prior to MCh challenge. Effect of GSNOR inhibitors arecompared to PBS vehicle dosed in the same manner.

Combivent is used as the positive control in all studies. Combivent(Boehringer Ingelheim) is administered to the lung using the inhalerdevice supplied with the product, but adapted for administration tomice, using a pipet tip. Combivent is administered 48 h, 24 h, and 1 hprior to MCh challenge. Each puff (or dose) of Combivent provides a doseof 18 μg ipatropium bromide (IpBr) and 103 μg albuterol sulfate orapproximately 0.9 mg/kg IpBr and 5 mg/kg albuterol.

Statistical Analyses

Area under the curve values for P_(enh) across baseline, saline, andincreasing doses of MCh challenge are calculated using GraphPad Prism5.0 (San Diego, Calif.) and expressed as a percent of the respective (IVor orally administered) vehicle control. Statistical differences amongtreatment groups and the respective vehicle control group within eachstudy are calculated using one-way ANOVA, Dunnetts (JMP 8.0, SASInstitute, Cary, N.C.). A p value of <0.05 among the treatment groupsand the respective vehicle control group is considered significantlydifferent.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention.

The invention claimed is:
 1. A compound of formula I:

wherein Ar₁ and Ar₂ are independently selected from the group consistingof aryl, substituted aryl, heteroaryl and substituted heteroaryl; X isselected from the group consisting of C or N; Y is C when X is N, and Nwhen X is C; and n is 0-3.
 2. The compound of claim 1 wherein: Ar₁ is asubstituted phenyl; and Ar₂ is selected from the group consisting ofphenyl, substituted phenyl, thiophen-yl, substituted thiophen-yl,pyridinyl, substituted pyridinyl, thiazolyl, substituted thiazolyl,bicyclic aryl, substituted bicyclic aryl, bicyclic heteroaryl,substituted bicyclic heteroaryl.
 3. The compound of claim 1 wherein: Ar₁is

wherein R₁ is selected from the group consisting of hydrogen, halogen,hydroxyl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro, CF₃, carbamoyl,C₁-C₆ alkylcarbamoyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆alkoxyl, and C₃-C₆ cycloalkoxyl; R₂ is selected from the groupconsisting of halogen, hydroxyl, carbamoyl, substituted carbamoyl, C₁-C₆alkylcarbamoyl, sulfamoyl, C₁-C₆ alkylsulfamoyl, C₁-C₆ alkyl, C₃-C₆cycloalkyl, cyano, nitro, amino, CF₃, carboxyl, ureido, sulfamoylamino,C₁-C₆ sulfonamido, 2-amino-2-oxoethyl, C₁-C₆ alkylamino, C₁-C₆dialkylamino, arylamino, heteroarylamino, C₁-C₆ alkoxyl, C₃-C₆cycloalkoxyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; R₃ is selected from the group consisting of hydrogen,hydroxyl, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, cyano, nitro,carbamoyl, C₁-C₆ alkylcarbamoyl, sulfamoyl, C₁-C₆ alkyl sulfamoyl,amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkoxyl, and C₃-C₆cycloalkoxyl; and Ar₂ is selected from the group consisting of phenyl,4-chlorophenyl, 3-chlorophenyl, 4-chloro-2-methoxyphenyl, 4-bromophenyl,4-bromo-2-methoxyphenyl, 3-bromophenyl, 4-fluorophenyl, 3-fluorophenyl,4-hydroxyphenyl, 4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl,4-chlorothiophen-2-yl, 5-chlorothiophen-2-yl, 3-bromothiophen-2-yl,4-bromothiophen-2-yl, 5-bromothiopheny-2-yl, 5-bromothiophen-3-yl,

wherein R₄ is selected from the group consisting of hydrogen, methyl,chloro, fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl,dimethylamino, amino, formamido, and trifluoromethyl R₅ is selected fromthe group consisting of hydrogen, methyl, and ethyl.
 4. The compound ofclaim 1 wherein Ar₂ is selected from the group consisting of phenyl,4-chloro-2-methoxyphenyl, 4-bromophenyl, 4-bromo-2-methoxyphenyl,4-methoxyphenyl, 4-(1H-imidazol-1-yl)phenyl,4-(2-methyl-1H-imidazol-1-yl)phenyl, and5-(2-methyl-1H-imidazol-1-yl)thiophen-2-yl.
 5. The compound of claim 3wherein R₁ is selected from the group consisting of hydrogen and methyl;R₂ is selected from the group consisting of hydroxyl, carboxyl,carbamoyl, methylsulfonamido, and tert-butyl-carboxy; and R₃ ishydrogen.
 6. The compound of claim 1 selected from the group consistingof: 4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-phenyl-1H-pyrrol-1-yl)benzamide;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-phenyl-1H-pyrrol-1-yl)benzoic acid;tert-butyl4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-phenyl-1H-pyrrol-1-yl)benzoate;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzamide;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol;4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)phenol;4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)phenol;4-(2-(4-(1H-imidazol-1-yl)phenyl)-5-(2-(1H-tetrazol-5-yl)ethyl)-1H-pyrrol-1-yl)phenol;4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzamide;4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzamide;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-bromophenyl)-1H-pyrrol-1-yl)-3-methylbenzamide;4-(2-(4-(1H-imidazol-1-yl)phenyl)-5-((1H-tetrazol-5-yl)methyl)-1H-pyrrol-1-yl)phenol;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-methoxyphenyl)-1H-pyrrol-1-yl)phenol;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-chloro-2-methoxyphenyl)-1H-pyrrol-1-yl)phenol;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-bromo-2-methoxyphenyl)-1H-pyrrol-1-yl)phenol;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-chloro-2-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzamide;4-(2-(2-(1H-tetrazol-5-yl)ethyl)-5-(4-bromo-2-methoxyphenyl)-1H-pyrrol-1-yl)-3-methylbenzamide;4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-1-yl)phenol;4-(2-((1H-tetrazol-5-yl)methyl)-5-(5-(2-methyl-1H-imidazol-1-yl)thiophen-2-yl)-1H-pyrrol-1-yl)-3-methylbenzamide;4-(2-((1H-tetrazol-5-yl)methyl)-5-(5-(2-methyl-1H-imidazol-1-yl)thiophen-2-yl)-1H-pyrrol-1-yl)phenol;4-(2-((1H-tetrazol-5-yl)methyl)-5-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-1H-pyrrol-1-yl)-3-methylbenzamide;N-(4-(2-((1H-tetrazol-5-yl)methyl)-5-(5-(2-methyl-1H-imidazol-1-yl)thiophen-2-yl)-1H-pyrrol-1-yl)-3-methylphenyl)methanesulfonamide;4-(1-(2-(1H-tetrazol-5-yl)ethyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide;and4-(1-((1H-tetrazol-5-yl)methyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl)-3-methylbenzamide.7. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound according to claim 1 together with apharmaceutically accepted carrier or excipient.